Cyanoaryl-aniline compounds for treatment of dermal disorders

ABSTRACT

Provided herein are compounds, pharmaceutical compositions comprising the compounds, methods of preparing the compounds, and methods of using the compounds and compositions in treating diseases or disorders in a subject where the subject is in need of an inhibitor of MEK where the compound is according to formula where R 1 , R 2 , R 2a , R 3 , R 3a , and X are as described herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application No. PCT/US2019/000068, filed Nov. 20, 2019,which claims priority to U.S. Provisional Application No. 62/769,871,filed Nov. 20, 2018, the contents of which are incorporated herein intheir entirety and for all purposes.

BACKGROUND OF THE INVENTION

Neurofibromatosis type 1 (NF1) occurs in approximately 1:3,500 births,and is one of the most common autosomal dominant single-gene disordersaffecting neurological function in humans. Clinically, NF1 disease ischaracterized by the presence of benign peripheral nerve tumors, calledneurofibromas, involving Schwann cells with biallelic mutations in theNF1 gene, as well as other tumor and non-tumor manifestations. SeeJousma et al. Pediatr. Blood Cancer 62: 1709-1716, 2015. NF1 isassociated with several dermal disorders, including dermalneurofibromas; plexiform neurofibromas; café au lait spots; and axillaryand inguinal freckling. Dermal neurofibromas occur in over 95% of NF1patients, and can appear anywhere on the body, causing itching,irritation, infection, physical pain, and disfigurement. Moreover,dermal neurofibromas are associated with social isolation and anxiety.

NF1 is caused by one or more germ line mutations in NF1, a gene thatinactivates the RAS pathway. Because the NF1 gene encodes a Ras-GAPprotein, NF1 loss results in high Ras-GTP. Therefore, NF1 research hasfocused intensively on testing inhibitors in the Ras signaling pathway,including the Ras-MAPK cascade. See Jousma et al. Pediatr. Blood Cancer62: 1709-1716, 2015. Four distinct MAPK cascades have been identifiedand named according to their MAPK module. See Akinleye et al. Journal ofHematology & Oncology 6:27, 2013. MEK proteins belong to a family ofenzymes that lie upstream to their specific MAPK targets in each of thefour MAP kinase signaling pathways. Two of these MEK proteins, MEK1 andMEK2, are closely related and participate in this signaling pathwaycascade. Inhibitors of MEK1 and MEK2 have been shown to effectivelyinhibit MEK signaling downstream of Ras, and thus provide a strongrationale for targeting MEK in the treatment of NFL. See Rice et al.Medicinal Chemistry Letters 3:416-421, 2012.

Currently available MEK inhibitors are designed to have oralbioavailability for systemic delivery, and are associated withsignificant side effects including decreased left ventricular ejectionfraction, elevated creatine phosphokinase, pneumonitis, renal failure,diarrhea, infection, uticaria, and maculo-papular rash, all of which aredose limiting or require permanent discontinuation. Moreover, clinicaltrials have shown side effects with prolonged high-dose administrationof MEK inhibitors. See Huang et al. J. Ocul. Pharmacol. Ther.25:519-530, 2009. For example, PD0325901, a MEK inhibitor currently inclinical trials, has exhibited neurological side effects associated withataxia, confusion, and syncope. In addition, a number of other sideeffects have been observed with systemic exposure to MEK inhibitorsincluding: acneiform rash, CPK elevation, nausea, vomiting, diarrhea,abdominal pain, and fatigue. Thus, there is a need for therapies thatinhibit MEK to treat NF1 associated dermal neurofibromas, which limitthese serious side effects.

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided herein is a compound of formula (I):

or a stereoisomer, mixture of stereoisomers, and/or a pharmaceuticallyacceptable salt thereof, wherein:

-   -   X is —CR^(3b) or N;    -   R¹ is —OR⁴, —NR⁵R^(5a), —N(OR^(5b))R^(5a), or a N-linked        heterocycloalkyl which is unsubstituted or substituted with one        or two R⁶;    -   R² is halo, C₁-C₆ alkyl, —S—C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₂-C₆        alkenyl, or C₂-C₆ alkynyl;    -   R^(2a) is halo or C₁-C₆ alkyl;    -   R³, R^(3a), and R^(3b) are each independently hydrogen, halo,        C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl;    -   R⁴, R⁵, and R^(5b) are each independently hydrogen, C₁-C₆ alkyl,        C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-C₁-C₆ alkyl, C₁-C₆        hydroxyalkyl, C₁-C₆ alkoxy-C₁-C₆ alkyl, amino-C₁-C₆ alkyl, C₁-C₆        alkylamino-C₁-C₆ alkyl, di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl,        heterocycloalkyl, heterocycloalkyl-C₁-C₆ alkyl, or R⁷—C(O)—C₁-C₆        alkyl, wherein each of the C₃-C₈ cycloalkyl and heterocycloalkyl        groups is unsubstituted or substituted with one to six R⁶;    -   R^(5a) is hydrogen or C₁-C₆ alkyl;    -   each R⁶ is independently halo, hydroxy, oxo, C₁-C₆ alkyl, C₃-C₈        cycloalkyl, C₁-C₆ alkoxy, C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl,        amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, amino-C₁-C₆        alkyl, C₁-C₆ alkylamino-C₁-C₆ alkyl, or di-(C₁-C₆        alkyl)amino-C₁-C₆ alkyl;    -   R⁷ is hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, di-(C₁-C₆        alkyl)amino, hydroxyamino, or N—C₁-C₆ alkyl hydroxyamino; and    -   provided that the compound is not ethyl        6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.

In a second aspect, provided herein is a pharmaceutical compositionincluding the compound of formula (I) and a pharmaceutically acceptablecarrier.

In a third aspect, provided herein is a method of treating aMEK-inhibitor responsive disorder, a MEK-inhibitor responsive dermaldisorder, a MEK-mediated disorder or disease, or a MEK-mediated dermaldisorder, the method including administering a therapeutically effectiveamount of a compound of formula (I) or a composition of the compound offormula (I) to a patient in need thereof, thereby treating the disorderor disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows synthesis Scheme 1 for the preparation of a compound offormula (Ia).

FIG. 2 shows synthesis Scheme 2 for the preparation of a compound offormula (Ia).

FIG. 3 shows synthesis Scheme 3 for the preparation of a compound offormula (Ib).

FIG. 4 shows suppression of pERK in human cutaneous neurofibromaexplants after treatment with Compound 1.030, using the protocoldescribed in Example 47.

FIG. 5 shows suppression of pERK in human cutaneous neurofibromaexplants after treatment with Compound 1.030 at a concentration of 500nM, using the protocol described in Example 47.

DETAILED DESCRIPTION OF THE INVENTION I. General

Provided herein are compounds of formula (I), pharmaceuticalcompositions including the compounds of formula (I), and methods ofusing these compounds or compositions in the treatment of aMEK-inhibitor responsive disorder or disease, a MEK-inhibitor responsivedermal disorder or disease, a MEK-mediated disorder or disease, or aMEK-mediated dermal disorder or disease.

II. Definition

The abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thesubstituents that would result from writing the structure from right toleft, e.g., —CH₂O— is meant to include —OCH₂—.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated (i.e., C₁-C₆ means one tosix carbons). Alkyl can include any number of carbons, such as C₁-C₂,C₁-C₃, C₁-C₄, C₁-C₅, C₁-C₆, C₁-C₇, C₁-C₈, C₁-C₉, C₁-C₁₀, C₂-C₃, C₂-C₄,C₂-C₅, C₂-C₆, C₃-C₄, C₃-C₅, C₃-C₆, C₄-C₅, C₄-C₆ and C₅-C₆. For example,C₁-C₆ alkyl includes, but is not limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbonsatoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated (i.e., C₁-C₆ meansone to six carbons), and linking at least two other groups, i.e., adivalent hydrocarbon radical. The two moieties linked to the alkylenecan be linked to the same atom or different atoms of the alkylene group.For instance, a straight chain alkylene can be the bivalent radical of—(CH₂)_(n)—, where n is 1, 2, 3, 4, 5 or 6. Representative alkylenegroups include, but are not limited to, methylene, ethylene, propylene,isopropylene, butylene, isobutylene, sec-butylene, pentylene andhexylene.

“Alkenyl” refers to a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond and having the numberof carbon atom indicated (i.e., C₂-C₆ means to two to six carbons).Alkenyl can include any number of carbons, such as C₂, C₂-C₃, C₂-C₄,C₂-C₅, C₂-C₆, C₂-C₇, C₂-C₈, C₂-C₉, C₂-C₁₀, C₃, C₃-C₄, C₃-C₅, C₃-C₆, C₄,C₄-C₅, C₄-C₅, C₅, C₅-C₆, and C₆. Alkenyl groups can have any suitablenumber of double bonds, including, but not limited to, 1, 2, 3, 4, 5 ormore. Examples of alkenyl groups include, but are not limited to, vinyl(ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.

“Alkynyl” refers to either a straight chain or branched hydrocarbonhaving at least 2 carbon atoms and at least one triple bond and havingthe number of carbon atom indicated (i.e., C₂-C₆ means to two to sixcarbons). Alkynyl can include any number of carbons, such as C₂, C₂-C₃,C₂-C₄, C₂-C₅, C₂-C₆, C₂-C₇, C₂-C₈, C₂-C₉, C₂-C₁₀, C₃, C₃-C₄, C₃-C₅,C₃-C₆, C₄, C₄-C₅, C₄-C₆, C₅, C₅-C₆, and C₆. Examples of alkynyl groupsinclude, but are not limited to, acetylenyl, propynyl, 1-butynyl,2-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl,1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or1,3,5-hexatriynyl.

“Cycloalkyl” refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃-C₆, C₄-C₆, C₅-C₆, C₃-C₈, C₄-C₈, C₅-C₈,C₆-C₈, C₃-C₉, C₃-C₁₀, C₃-C₁₁, and C₃-C₁₂. Saturated monocycliccycloalkyl rings include, for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic andpolycyclic cycloalkyl rings include, for example, norbornane,[2.2.2]bicyclooctane, decahydronaphthalene and adamantane. Cycloalkylgroups can also be partially unsaturated, having one or more double ortriple bonds in the ring. Representative cycloalkyl groups that arepartially unsaturated include, but are not limited to, cyclobutene,cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers),cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-and 1,5-isomers), norbornene, and norbornadiene. When cycloalkyl is asaturated monocyclic C₃-C₈ cycloalkyl, exemplary groups include, but arenot limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

“Cycloalkylalkyl” refers to a radical having an alkyl component and acycloalkyl component, where the alkyl component links the cycloalkylcomponent to the point of attachment. The alkyl component is as definedabove, except that the alkyl component is at least divalent, analkylene, to link to the cycloalkyl component and to the point ofattachment. The alkyl component can include any number of carbons, suchas C₁-C₆, C₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, C₃-C₄,C₃-C₅, C₃-C₆, C₄-C₅, C₄-C₆ and C₅-C₆. The cycloalkyl component is asdefined above. Exemplary cycloalkyl-alkyl groups include, but are notlimited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl andcyclohexylmethyl.

“Alkoxy” refers to an alkyl group having an oxygen atom that connectsthe alkyl group to the point of attachment: alkyl-O—. Alkoxy groups canhave any suitable number of carbon atoms, such as C₁-C₆. Alkoxy groupsinclude, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.

“Hydroxyalkyl” refers to an alkyl group, as defined above, where atleast one of the hydrogen atoms is replaced with a hydroxy group. As forthe alkyl group, a hydroxyalkyl group can have any suitable number ofcarbon atoms, such as C₁-C₆. Exemplary hydroxyalkyl groups include, butare not limited to, hydroxymethyl, hydroxyethyl (where the hydroxy is inthe 1- or 2-position), hydroxypropyl (where the hydroxy is in the 1-, 2-or 3-position), hydroxybutyl (where the hydroxy is in the 1-, 2-, 3- or4-position), hydroxypentyl (where the hydroxy is in the 1-, 2-, 3-, 4-or 5-position), hydroxyhexyl (where the hydroxy is in the 1-, 2-, 3-,4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like.

“Alkoxyalkyl” refers to a radical having an alkyl component and analkoxy component, where the alkyl component links the alkoxy componentto the point of attachment. The alkyl component is as defined above,except that the alkyl component is at least divalent, an alkylene, tolink to the alkoxy component and to the point of attachment. The alkylcomponent can include any number of carbons, such as C₁-C₂, C₁-C₃,C₁-C₄, C₁-C₅, C₁-C₆, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, C₃-C₄, C₃-C₅, C₃-C₆,C₄-C₅, C₄-C₆ and C₅-C₆. The alkoxy component is as defined above.Examples of the alkoxy-alkyl group include, but are not limited to,2-ethoxy-ethyl and methoxymethyl.

“Halogen” or “halo” refers to fluoro, chloro, bromo, or iodo.

“Haloalkyl” refers to alkyl, as defined above, where some or all of thehydrogen atoms are replaced with halogen atoms. As for alkyl group,haloalkyl groups can have any suitable number of carbon atoms, such asC₁-C₆. For example, haloalkyl includes trifluoromethyl, fluoromethyl,2,2,2-trifluoroethyl, etc. In some instances, the term “perfluoro” canbe used to define a compound or radical where all the hydrogens arereplaced with fluorine. For example, perfluoromethyl refers to1,1,1-trifluoromethyl.

“Amino” as used herein, and unless otherwise specified, refers to —NH₂.

“Alkylamino” as used herein, and unless otherwise specified, refers toan —NHR radical where R is alkyl as defined herein, or an N-oxidederivative thereof. In some embodiments, alkylamino is C₁-C₆ alkylamino.In some embodiments, C₁-C₆ alkylamino is methylamino, ethylamino, n-,iso-propylamino, n-, iso-, tert-butylamino, or methylamino-N-oxide, andthe like.

“Dialkylamino” as used herein, and unless otherwise specified, refers toan —NR′R radical where R and R′ are independently alkyl as definedherein, or an N-oxide derivative thereof. In some embodiments,dialkylamino is di-(C₁-C₆ alkyl)amino. In some embodiments, di-(C₁-C₆alkyl)amino is dimethylamino, methyl-ethylamino, diethylamino, ordimethylamino-N-oxide, and the like.

“Aminoalkyl” as used herein, unless otherwise specified, refers to analkyl group substituted with one or two NH₂. In some embodiments,aminoalkyl is amino-C₁-C₆ alkyl.

“Alkylaminoalkyl” as used herein, unless otherwise specified, refers toan alkyl group substituted with one or two —NH(alkyl) groups. In someembodiments, alkylaminoalkyl is C₁-C₆ alkylamino-C₁-C₆ alkyl.

“Dialkylaminoalkyl” as used herein, unless otherwise specified, refersto an alkyl group substituted with one or two —N(alkyl)₂ groups. In someembodiments, dialkylaminoalkyl is di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl.

“Hydroxyamino” as used herein, unless otherwise specified, refers to—NHOH.

“N-alkylhydroxyamino” as used herein, unless otherwise specified, refersto the amine hydrogen of —NHOH is substituted with alkyl as definedherein. In some embodiments, N-alkyl hydroxyamino is N—C₁-C₆alkyl-hydroxyamino. In some embodiments, N—C₁-C₆ alkyl-hydroxyamino isN-methylhydroxyamino, N-ethylhydroxyamino, N-(n-,iso-propyl)-hydroxyamino, or N-(n-, iso-, tert-butyl)hydroxyamino, andthe like.

“Heterocycloalkyl” refers to a saturated ring system having from 3 to 12ring members and from 1 to 4 heteroatoms selected from N, O and S. Theheteroatoms can also be oxidized, such as, but not limited to, —S(O)—and —S(O)₂—. Heterocycloalkyl groups can include any number of ringatoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8,3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable numberof heteroatoms can be included in the heterocycloalkyl groups, such as1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Theheterocycloalkyl group can include groups such as aziridinyl,azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, azocanyl,quinuclidinyl, pyrazolidinyl, imidazolidinyl, piperazinyl (1,2-, 1,3-and 1,4-isomers), oxiranyl, oxetanyl, tetrahydrofuranyl, oxanyl(tetrahydropyranyl), oxepanyl, thiiranyl, thietanyl, thiolanyl(tetrahydrothiophenyl), thianyl (tetrahydrothiopyranyl), oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl,dithiolanyl, morpholinyl, thiomorpholinyl, dioxanyl, or dithianyl. Theheterocycloalkyl groups can also be fused to aromatic or non-aromaticring systems to form members including, but not limited to, indoline.Heterocycloalkyl groups can be unsubstituted or substituted. Forexample, heterocycloalkyl groups can be substituted with C₁-C₆ alkyl oroxo (═O), among many others.

The heterocycloalkyl groups can be linked via any position on the ring.For example, aziridinyl can be 1- or 2-aziridinyl, azetidinyl can be 1-or 2-azetidinyl, pyrrolidinyl can be 1-, 2- or 3-pyrrolidinyl,piperidinyl can be 1-, 2-, 3- or 4-piperidinyl, pyrazolidinyl can be 1-,2-, 3-, or 4-pyrazolidinyl, imidazolidinyl can be 1-, 2-, 3- or4-imidazolidinyl, piperazinyl can be 1-, 2-, 3- or 4-piperazinyl,tetrahydrofuranyl can be 1- or 2-tetrahydrofuranyl, oxazolidinyl can be2-, 3-, 4- or 5-oxazolidinyl, isoxazolidinyl can be 2-, 3-, 4- or5-isoxazolidinyl, thiazolidinyl can be 2-, 3-, 4- or 5-thiazolidinyl,isothiazolidinyl can be 2-, 3-, 4- or 5-isothiazolidinyl, andmorpholinyl can be 2-, 3- or 4-morpholinyl.

“N-linked heterocycloalkyl” or “nitrogen-linked heterocycloalkyl” refersto the heterocycloalkyl group linked via N-position on the ring. Forexample, N-linked aziridinyl is aziridin-1-yl, N-linked azetidinyl isazetidin-1-yl, N-linked pyrrolidinyl is pyrrolidin-1-yl, N-linkedpiperidinyl is piperidin-1-yl, N-linked pyrazolidinyl ispyrazolidin-1-yl or pyrazolidin-2-yl, N-linked imidazolidinyl can beimidazolidin-1-yl or imidazolidin-3-yl, N-linked piperazinyl ispiperazin-1-yl or piperazin-4-yl, N-linked oxazolidinyl isoxazolidin-3-yl, N-linked isoxazolidinyl is isoxazolidin-2-yl, N-linkedthiazolidinyl is thiazolidin-3-yl, N-linked isothiazolidinyl isisothiazolidin-2-yl, and N-linked morpholinyl is 4-morpholinyl.

When heterocycloalkyl includes 3 to 8 ring members and 1 to 3heteroatoms, representative members include, but are not limited to,pyrrolidinyl, piperidinyl, tetrahydrofuranyl, oxanyl,tetrahydrothiophenyl, thianyl, pyrazolidinyl, imidazolidinyl,piperazinyl, oxazolidinyl, isoxzoalidinyl, thiazolidinyl,isothiazolidinyl, morpholinyl, thiomorpholinyl, dioxanyl and dithianyl.Heterocycloalkyl can also form a ring having 5 to 6 ring members and 1to 2 heteroatoms, with representative members including, but not limitedto, pyrrolidinyl, piperidinyl, tetrahydrofuranyl, tetrahydrothiophenyl,pyrazolidinyl, imidazolidinyl, piperazinyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, and morpholinyl.

“Protecting group” refers to a compound that renders a functional groupunreactive to a particular set of reaction conditions, but that is thenremovable in a later synthetic step so as to restore the functionalgroup to its original state. Such protecting groups are well known toone of ordinary skill in the art and include compounds that aredisclosed in “Protective Groups in Organic Synthesis”, 4th edition, T.W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which isincorporated herein by reference in its entirety.

“Salt” refers to acid or base salts of the compounds of the presentinvention. Illustrative examples of pharmaceutically acceptable saltsare mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid,and the like) salts, organic acid (acetic acid, propionic acid, glutamicacid, citric acid and the like) salts, quaternary ammonium (methyliodide, ethyl iodide, and the like) salts. It is understood that thepharmaceutically acceptable salts are non-toxic. Additional informationon suitable pharmaceutically acceptable salts can be found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, which is incorporated herein by reference.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

“Isomer” refers to compounds with the same chemical formula but whichare structurally distinguishable. Certain compounds of the presentinvention possess asymmetric carbon atoms (optical centers) or doublebonds; the racemates, diastereomers, geometric isomers and individualisomers are all intended to be encompassed within the scope of thepresent invention.

“Tautomer” refers to one of two or more structural isomers which existin equilibrium and which are readily converted from one form to another.

“Solvate” refers to a compound provided herein or a salt thereof, thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

“Hydrate” refers to a compound that is complexed to at least one watermolecule. The compounds of the present invention can be complexed withfrom 1 to 10 water molecules.

“Substantially free of” or “substantially in the absence of”stereoisomers with respect to a composition refers to a composition thatincludes at least 85 or 90% by weight, in some embodiments 95%, 98%, 99%or 100% by weight, of a designated stereoisomer of a compound in thecomposition. In some embodiments, in the methods and compounds providedherein, the compounds are substantially free of stereoisomers.

“Isolated” with respect to a composition refers to a composition thatincludes at least 85%, 90%, 95%, 98%, 99% to 100% by weight, of aspecified compound, the remainder comprising other chemical species orstereoisomers.

“Composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product, which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and deleterious to the recipient thereof.

“Pharmaceutically acceptable excipient” refers to a substance that aidsthe administration of an active agent to and absorption by a subject.Pharmaceutical excipients useful in the present invention include, butare not limited to, binders, fillers, disintegrants, lubricants,coatings, sweeteners, flavors and colors. One of skill in the art willrecognize that other pharmaceutical excipients are useful in the presentinvention.

“IC₅₀” refers to an amount, concentration or dosage of a particular testcompound that achieves a 50% inhibition of a maximal response in anassay that measures such response.

“Inhibition”, “inhibits” and “inhibitor” refer to a compound thatprohibits or a method of prohibiting, a specific action or function.

“Administering” refers to oral administration, administration as asuppository, topical contact, parenteral, intravenous, intraperitoneal,intramuscular, intralesional, intranasal or subcutaneous administration,intrathecal administration, or the implantation of a slow-release devicee.g., a mini-osmotic pump, to the subject.

“Treat”, “treating” and “treatment” refer to any indicia of success inthe treatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the injury, pathology orcondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; improving a patient's physical or mental well-being. Thetreatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation.

“Patient” or “subject” refers to a living organism suffering from orprone to a disease or condition that can be treated by administration ofa pharmaceutical composition as provided herein. Non-limiting examplesinclude humans, other mammals, bovines, rats, mice, dogs, monkeys, goat,sheep, cows, deer, and other non-mammalian animals. In some embodiments,the patient is human.

“Therapeutically effective amount” refers to an amount of a compound orof a pharmaceutical composition useful for treating or ameliorating anidentified disease or condition, or for exhibiting a detectabletherapeutic or inhibitory effect. The exact amounts will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Pickar, Dosage Calculations(1999); and Remington: The Science and Practice of Pharmacy, 20thEdition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

The disclosure provides “soft” MEK inhibitors, compositions comprising“soft” MEK inhibitors, and methods of treating and/or preventing adermal disorder (e.g., a MEK-inhibitor responsive dermal disorder or aMEK mediated dermal disorder, e.g., a dermal rasopathy, e.g., a dermaldisorder associated with neurofibromatosis type 1 (NF1), e.g., a dermalneurofibroma, a subdermal neurofibroma, or a superficial plexiformneurofibroma) with MEK inhibitors e.g., “soft” MEK inhibitors. Forexample, the methods described herein provide administration, e.g.,local or non-systemic, e.g., topical, intradermal, or intralesionaladministration, of MEK inhibitors, e.g., “soft” MEK inhibitors, e.g.,“soft” MEK inhibitors described herein, whereby the side effectsexhibited with systemic exposure, e.g., known side effects exhibitedwith MEK inhibitors designed for systemic delivery, are significantlyreduced.

In some embodiments, “soft MEK inhibitor” is a compound which inhibitsMEK1 and/or 2 and is characterized by a predictable and controllablemetabolism/degradation to non-toxic and biologically less active orinactive (i.e. does not inhibit, or inhibits to a lesser degree, MEK1and/or 2) products after they have achieved their therapeutic role inthe skin.

“Hard MEK inhibitor” refers to a MEK inhibitor known in the art. In someembodiments, a hard MEK inhibitor is designed for oral bioavailability.This is necessary to deliver therapeutically effective levels of MEKinhibitor to peripheral lesions with systemic delivery. Hard MEKinhibitor include, for example, PD0325901; PD184161; SMK-17; AS703026(Pimasertib, MSC1936369); RO-4987655; Selumetinib (AZD6244, ARRY142886);Binimetinib (MEK162, ARRY-162, ARRY-438162); Refametinib; Cobimetinib(GDC-0973, XL518); GDC-0623; AZD8330 (ARRY-424704); CI-1040 (PD184352);PD198306; and PD318088.

While not wishing to be bound by theory, it is believed that soft MEKinhibitors, e.g., such as the “soft” MEK inhibitors described herein,are more metabolically labile than known “hard” MEK inhibitors. Due totheir inherent metabolic instability, e.g., for degradation uponreaching the systemic circulation, “soft” MEK inhibitors, e.g., such asthe “soft” MEK inhibitors described herein, are dermally active but havelow systemic exposure upon local or non-systemic administration, e.g.,topical, intradermal, or intralesional administration, because theyrapidly degrade upon exposure to plasma or blood or hepatic metabolicenzymes. Unlike “soft” MEK inhibitors, known MEK inhibitors have beenhistorically designed for oral bioavailability, which requires goodstability in plasma or blood and good stability to hepatic metabolismnecessary to permit systemic delivery at therapeutically effectivelevels, and are more prone to unwanted side effects and increasedtoxicity. As a result, “soft” MEK inhibitors, e.g., such as the soft MEKinhibitors described herein, are less systemically toxic.

“A,” “an,” or “a(n)”, when used in reference to a group of substituentsor “substituent group” herein, mean at least one. For example, where acompound is substituted with “an” alkyl or aryl, the compound isoptionally substituted with at least one alkyl and/or at least one aryl,wherein each alkyl and/or aryl is optionally different. In anotherexample, where a compound is substituted with “a” substitutent group,the compound is substituted with at least one substituent group, whereineach substitutent group is optionally different.

III. Compounds

In one aspect, provided herein is a compound of formula (I):

or a stereoisomer, mixture of stereoisomers, and/or a pharmaceuticallyacceptable salt thereof, wherein:

-   -   X is —CR^(3b) or N;    -   R¹ is —OR⁴, —NR⁵R^(5a), —N(OR^(5b))R^(5a), or a N-linked        heterocycloalkyl which is unsubstituted or substituted with one        or two R⁶;    -   R² is halo, C₁-C₆ alkyl, —S—C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₂-C₆        alkenyl, or C₂-C₆ alkynyl;    -   R^(2a) is halo or C₁-C₆ alkyl;    -   R³, R^(3a), and R^(3b) are each independently hydrogen, halo,        C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl;    -   R⁴, R⁵, and R^(5b) are each independently hydrogen, C₁-C₆ alkyl,        C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-C₁-C₆ alkyl, C₁-C₆        hydroxyalkyl, C₁-C₆ alkoxy-C₁-C₆ alkyl, amino-C₁-C₆ alkyl, C₁-C₆        alkylamino-C₁-C₆ alkyl, di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl,        heterocycloalkyl, heterocycloalkyl-C₁-C₆ alkyl, or R⁷—C(O)—C₁-C₆        alkyl, wherein each of the C₃-C₈ cycloalkyl and heterocycloalkyl        groups is unsubstituted or substituted with one to six R⁶;    -   R^(5a) is hydrogen or C₁-C₆ alkyl;    -   each R⁶ is independently halo, hydroxy, oxo, C₁-C₆ alkyl, C₃-C₈        cycloalkyl, C₁-C₆ alkoxy, C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl,        amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, amino-C₁-C₆        alkyl, C₁-C₆ alkylamino-C₁-C₆ alkyl, or di-(C₁-C₆        alkyl)amino-C₁-C₆ alkyl;    -   R⁷ is hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, di-(C₁-C₆        alkyl)amino, hydroxyamino, or N—C₁-C₆ alkyl hydroxyamino; and    -   provided that the compound is not ethyl        6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.

In some embodiments, the cycloalkyl group of R², R⁴, R⁵, R^(5b) and R⁶is a saturated monocyclic C₃-C₈ cycloalkyl. In some embodiments, theC₃-C₈ cycloalkyl group, as alone or as part of C₃-C₈ cycloalkyl-C₁-C₆alkyl is cyclopropyl or cyclobutyl. In some embodiments, the C₃-C₈cycloalkyl group, as alone or as part of C₃-C₈ cycloalkyl-C₁-C₆ alkyl,is unsubstituted. In some embodiments, the C₃-C₈ cycloalkyl group, asalone or as part of C₃-C₈ cycloalkyl-C₁-C₆ alkyl, is substituted withone to six R⁶ and R⁶ is as defined and described herein.

With reference to R⁶ as one or more substituents of the C₃-C₈ cycloalkylgroup, in some embodiments, each R⁶ is independently halo, hydroxy, oxo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl, amino,C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, amino-C₁-C₆ alkyl, C₁-C₆alkylamino-C₁-C₆ alkyl, or di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl. In someembodiments, each R⁶ is independently halo, hydroxy, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl, amino, C₁-C₆ alkylamino, ordi-(C₁-C₆ alkyl)amino. In some embodiments, each R⁶ is independentlyhalo, hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, or amino. In some embodiments,each R⁶ is independently hydroxy or C₁-C₆ alkyl. In some embodiments,each R⁶ is independently hydroxy or amino.

In some embodiments, heterocycloalkyl of R¹, R⁴, R⁵ and R^(5b) is a 3 to8 membered heterocycloalkyl having 1 to 3 heteroatoms of N, O, or S. Insome embodiments, heterocycloalkyl is a 3 to 6 membered heterocycloalkylhaving 1 to 2 heteroatoms of N or O. In some embodiments, theheterocycloalkyl group, as alone or as part of heterocycloalkyl-C₁-C₆alkyl, is unsubstituted. In some embodiments, the heterocycloalkylgroup, as alone or as part of heterocycloalkyl-C₁-C₆ alkyl, issubstituted one to six R⁶ and R⁶ is as defined and described herein. Insome embodiments, the N-linked heterocycloalkyl group is substituted oneor two R⁶ and R⁶ is as defined and described herein.

With reference to R⁶ as one or more substituents of the heterocycloalkylgroup or the N-linked heterocycloalkyl, in some embodiments, each R⁶ isindependently halo, hydroxy, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy,C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl, amino, C₁-C₆ alkylamino, di-(C₁-C₆alkyl)amino, amino-C₁-C₆ alkyl, C₁-C₆ alkylamino-C₁-C₆ alkyl, ordi-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl. In some embodiments, each R⁶ isindependently halo, hydroxy, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy,C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl, amino, C₁-C₆ alkylamino, ordi-(C₁-C₆ alkyl)amino. In some embodiments, each R⁶ is independentlyhalo, hydroxy, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, or amino. In someembodiments, each R⁶ is independently hydroxy or C₁-C₆ alkyl. In someembodiments, each R⁶ is independently hydroxy, oxo, or amino. In someembodiments, each R⁶ is independently hydroxy or amino.

In some embodiments, X is —CR^(3b), and the compound is represented byformula (Ia):

wherein R¹, R², R^(2a), R³, R^(3a), and R^(3b) are as defined herein inany aspect or embodiment described herein.

In some embodiments of formula (Ia), R³, R^(3a), and R^(3b) are eachindependently hydrogen, halo, or C₁-C₆ alkyl.

In some embodiments of formula (Ia), R³ is hydrogen, halo, or C₁-C₆alkyl. In some embodiments, R³ is hydrogen. In some embodiments, R³ ishalo. In some embodiments, R³ is fluoro, chloro, bromo, or iodo. In someembodiments, R³ is fluoro. In some embodiments, R³ is C₁-C₆ alkyl. Insome embodiments, R³ is methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, or hexyl. In someembodiments, R³ is methyl.

In some embodiments of formula (Ia), R^(3a) is hydrogen, halo, or C₁-C₆alkyl. In some embodiments, R^(3a) is hydrogen. In some embodiments,R^(3a) is halo. In some embodiments, R^(3a) is fluoro, chloro, bromo, oriodo. In some embodiments, R^(3a) is fluoro. In some embodiments, R^(3a)is C₁-C₆ alkyl. In some embodiments, R^(3a) is methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, orhexyl. In some embodiments, R^(3a) is methyl.

In some embodiments of formula (Ia), R^(3b) is hydrogen, halo, or C₁-C₆alkyl. In some embodiments, R^(3b) is hydrogen. In some embodiments,R^(3b) is halo. In some embodiments, R^(3b) is fluoro, chloro, bromo, oriodo. In some embodiments, R^(3b) is fluoro. In some embodiments, R^(3b)is C₁-C₆ alkyl. In some embodiments, R^(3b) is methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, orhexyl. In some embodiments, R^(3b) is methyl.

In some embodiments of formula (Ia), R³, R^(3a), and R^(3b) are eachhydrogen. In some embodiments of formula (Ia), R³ and R^(3a) are eachhydrogen and R^(3b) is halo. In some embodiments of formula (Ia), R³ andR^(3a) are each hydrogen and R^(3b) is fluoro.

Returning to formula (I), in some embodiments, X is N, and the compoundis represented by formula (Ib):

wherein R¹, R², R^(2a), R³, and R^(3a) are as defined herein in anyaspect or embodiment described herein.

In some embodiments of formula (Ib), R³ and R^(3a) are eachindependently hydrogen, halo, or C₁-C₆ alkyl.

In some embodiments of formula (Ib), R³ is hydrogen, halo, or C₁-C₆alkyl. In some embodiments, R³ is hydrogen. In some embodiments, R³ ishalo. In some embodiments, R³ is fluoro, chloro, bromo, or iodo. In someembodiments, R³ is fluoro. In some embodiments, R³ is C₁-C₆ alkyl. Insome embodiments, R³ is methyl.

In some embodiments of formula (Ib), R^(3a) is hydrogen, halo, or C₁-C₆alkyl. In some embodiments, R^(3a) is hydrogen. In some embodiments,R^(3a) is halo. In some embodiments, R^(3a) is fluoro, chloro, bromo, oriodo. In some embodiments, R^(3a) is fluoro. In some embodiments, R^(3a)is C₁-C₆ alkyl. In some embodiments, R^(3a) is methyl.

In some embodiments of formula (Ib), R³ and R^(3a) are eachindependently hydrogen or halo. In some embodiments, R³ and R^(3a) areeach hydrogen. In some embodiments, one of R³ and R^(3a) is hydrogen andthe other is halo. In some embodiments, one of R³ and R^(3a) is hydrogenand the other is fluoro.

With reference to any one of formulae (I), (Ia), and (Ib), in someembodiments, R² is halo, C₁-C₆ alkyl, —S—C₁-C₆ alkyl, C₃-C₈ cycloalkyl,C₂-C₆ alkenyl, or C₂-C₆ alkynyl. In some embodiments, R² is halo orC₁-C₆ alkyl. In some embodiments, R² is halo, —SCH₃, —CH₃, C₂-C₃alkenyl, or C₂-C₃ alkynyl.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R² ishalo. In some embodiments, R² is fluoro. In some embodiments, R² isiodo. In some embodiments, R² is chloro. In some embodiments, R² isbromo.

In some embodiments of any one of formulae (I), (la), and (Ib), R² isC₁-C₆ alkyl. In some embodiments, R² is C₁-C₃ alkyl. In someembodiments, R² is methyl.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R² is—S—C₁-C₆ alkyl. In some embodiments, R² is —S—C₁-C₃ alkyl. In someembodiments, R² is —SCH₃.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R² isC₃-C₈ cycloalkyl. In some embodiments, R² is cyclopropyl.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R² isC₂-C₆ alkenyl. In some embodiments, R² is C₂-C₄ alkenyl. In someembodiments, R² is vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl,2-butenyl, isobutenyl, or butadienyl. In some embodiments, R² is vinyl.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R² isC₂-C₆ alkynyl. In some embodiments, R² is C₂-C₃ alkynyl. In someembodiments, R² is acetylenyl or propynyl. In some embodiments, R² isacetylenyl.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R^(2a)is halo or C₁-C₃ alkyl. In some embodiments, R^(2a) is halo or CH₃. Insome embodiments, R^(2a) is fluoro or CH₃. In some embodiments, R^(2a)is iodo or CH₃. In some embodiments, R^(2a) is chloro or CH₃. In someembodiments, R^(2a) is bromo or CH₃.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R^(2a)is halo. In some embodiments, R^(2a) is fluoro. In some embodiments,R^(2a) is iodo. In some embodiments, R^(2a) is chloro. In someembodiments, R^(2a) is bromo.

In some embodiments of any one of formulae (I), (la), and (Ib), R^(2a)is C₁-C₆ alkyl. In some embodiments, R^(2a) is C₁-C₃ alkyl. In someembodiments, R^(2a) is CH₃.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R² andR^(2a) are each halo. In some embodiments, R² is halo and R^(2a) isC₁-C₆ alkyl. In some embodiments, R² is C₁-C₆ alkyl and R^(2a) is halo.In some embodiments, R² is —S—C₁-C₆ alkyl and R^(2a) is halo. In someembodiments, R² is —SCH₃ and R^(2a) is halo. In some embodiments, R² isC₃-C₈ cycloalkyl and R^(2a) is halo. In some embodiments, R² iscyclopropyl and R^(2a) is halo. In some embodiments, R² is C₂-C₆ alkenyland R^(2a) is halo. In some embodiments, R² is C₂-C₆ alkynyl and R^(2a)is halo. In some embodiments, R² is acetylenyl and R^(2a) is halo. Insome embodiments, R² and R^(2a) are each independently fluoro, chloro,bromo, or iodo. In some embodiments, R² is iodo and R^(2a) is fluoro. Insome embodiments, R² is halo and R^(2a) is —CH₃. In some embodiments, R²is bromo and R^(2a) is —CH₃. In some embodiments, R² is iodo and R^(2a)is —CH₃. In some embodiments, R² is —SCH₃ and R^(2a) is fluoro. In someembodiments, R² is acetylenyl and R^(2a) is fluoro.

With reference to any one of formulae (I), (la), and (Ib), in someembodiments, R¹ is —OR⁴, —NR⁵R^(5a), or —N(OR^(5b))R^(5a)

In some embodiments of any one of formulae (I), (la), and (Ib), R¹ is—OR⁴. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ isC₁-C₆ alkyl. In some embodiments, R⁴ is C₃-C₈ cycloalkyl unsubstitutedor substituted with one to six R⁶ and R⁶ is as defined and describedherein. In some embodiments, R⁴ is C₃-C₈ cycloalkyl unsubstituted orsubstituted with one to six R⁶ and each R⁶ is independently hydroxy orC₁-C₆ alkyl. In some embodiments, R⁴ is C₃-C₈ cycloalkyl-C₁-C₆ alkyl,wherein the C₃-C₈ cycloalkyl group is unsubstituted or substituted withone to six R⁶ and R⁶ is as defined and described herein. In someembodiments, R⁴ is C₃-C₈ cycloalkyl-C₁-C₆ alkyl, wherein the C₃-C₈cycloalkyl group is unsubstituted or substituted with one to six R⁶ andeach R⁶ is independently hydroxy or C₁-C₆ alkyl. In some embodiments, R⁴is cyclopropyl, cyclobutyl, cyclopropyl-C₁-C₆ alkyl, or cyclobutyl-C₁-C₆alkyl; and each of the cyclopropyl and cyclobutyl groups isunsubstituted or substituted with one to six R⁶ and R⁶ is as defined anddescribed herein. In some embodiments, R⁴ is cyclopropyl, cyclobutyl,cyclopropyl-C₁-C₆ alkyl, or cyclobutyl-C₁-C₆ alkyl; and each of thecyclopropyl and cyclobutyl groups is unsubstituted or substituted withone to six R⁶ and each R⁶ is independently hydroxy or C₁-C₆ alkyl. Insome embodiments, R⁴ is C₁-C₆ hydroxyalkyl. In some embodiments, R⁴ isC₁-C₆ alkoxy-C₁-C₆ alkyl. In some embodiments, R⁴ is amino-C₁-C₆ alkyl.In some embodiments, R⁴ is C₁-C₆ alkylamino-C₁-C₆ alkyl. In someembodiments, R⁴ is di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl. In someembodiments, R⁴ is heterocycloalkyl unsubstituted or substituted withone to six R⁶ and R⁶ is as defined and described herein. In someembodiments, R⁴ is heterocycloalkyl unsubstituted or substituted withone to six R⁶ and each R⁶ is independently hydroxy or C₁-C₆ alkyl. Insome embodiments, R⁴ is heterocycloalkyl-C₁-C₆ alkyl, wherein theheterocycloalkyl group is unsubstituted or substituted with one to sixR⁶ and R⁶ is as defined and described herein. In some embodiments, R⁴ isheterocycloalkyl-C₁-C₆ alkyl, wherein the heterocycloalkyl group isunsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl. In some embodiments, R⁴ isoxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, oxetanyl-C₁-C₆ alkyl,azetidinyl-C₁-C₆ alkyl, pyrrolidinyl-C₁-C₆ alkyl, piperidinyl-C₁-C₆alkyl, or 2,2-dimethyl-1,3-dioxolan-4-yl-C₁-C₆ alkyl. In someembodiments, R⁴ is R⁷—C(O)—C₁-C₆ alkyl; and R⁷ is hydroxy, C₁-C₆ alkoxy,amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, hydroxyamino, or N—C₁-C₆alkyl hydroxyamino. In some embodiments, R⁴ is R⁷—C(O)—C₁-C₆ alkyl; andR⁷ is hydroxy, C₁-C₆ alkoxy, amino, or hydroxyamino.

In some embodiments of any one of formulae (I), (la), and (Ib), R¹ isselected from the group consisting of —OH,

In some embodiments of any one of formulae (I), (Ia), and (Ib), R¹ is—NR⁵R^(5a). In some embodiments, R⁵ is hydrogen. In some embodiments, R⁵is C₁-C₆ alkyl. In some embodiments, R⁵ is C₃-C₈ cycloalkylunsubstituted or substituted with one to six R⁶ and R⁶ is as defined anddescribed herein. In some embodiments, R⁵ is C₃-C₈ cycloalkylunsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl. In some embodiments, R⁵ is C₃-C₈cycloalkyl-C₁-C₆ alkyl, wherein the C₃-C₈ cycloalkyl group isunsubstituted or substituted with one to six R⁶ and R⁶ is as defined anddescribed herein. In some embodiments, R⁵ is C₃-C₈ cycloalkyl-C₁-C₆alkyl, wherein the C₃-C₈ cycloalkyl group is unsubstituted orsubstituted with one to six R⁶ and each R⁶ is independently hydroxy orC₁-C₆ alkyl. In some embodiments, R⁵ is cyclopropyl, cyclobutyl,cyclopropyl-C₁-C₆ alkyl, or cyclobutyl-C₁-C₆ alkyl; and each of thecyclopropyl and cyclobutyl groups is unsubstituted or substituted withone to six R⁶ and R⁶ is as defined and described herein. In someembodiments, R⁵ is cyclopropyl, cyclobutyl, cyclopropyl-C₁-C₆ alkyl, orcyclobutyl-C₁-C₆ alkyl; and each of the cyclopropyl and cyclobutylgroups is unsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl. In some embodiments, R⁵ is C₁-C₆hydroxyalkyl. In some embodiments, R⁵ is C₁-C₆ alkoxy-C₁-C₆ alkyl. Insome embodiments, R⁵ is amino-C₁-C₆ alkyl. In some embodiments, R⁵ isC₁-C₆ alkylamino-C₁-C₆ alkyl. In some embodiments, R⁵ is di-(C₁-C₆alkyl)amino-C₁-C₆ alkyl. In some embodiments, R⁵ is heterocycloalkylunsubstituted or substituted with one to six R⁶ and R⁶ is as defined anddescribed herein. In some embodiments, R⁵ is heterocycloalkylunsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl. In some embodiments, R⁵ isheterocycloalkyl-C₁-C₆ alkyl, wherein the heterocycloalkyl group isunsubstituted or substituted with one to six R⁶ and R⁶ is as defined anddescribed herein. In some embodiments, R⁵ is heterocycloalkyl-C₁-C₆alkyl, wherein the heterocycloalkyl group is unsubstituted orsubstituted with one to six R⁶ and each R⁶ is independently hydroxy orC₁-C₆ alkyl. In some embodiments, R⁵ is oxetanyl, azetidinyl,pyrrolidinyl, piperidinyl, oxetanyl-C₁-C₆ alkyl, azetidinyl-C₁-C₆ alkyl,pyrrolidinyl-C₁-C₆ alkyl, piperidinyl-C₁-C₆ alkyl, or2,2-dimethyl-1,3-dioxolan-4-yl-C₁-C₆ alkyl. In some embodiments, R⁵ isR⁷—C(O)—C₁-C₆ alkyl; and R⁷ is hydroxy, C₁-C₆ alkoxy, amino, C₁-C₆alkylamino, di-(C₁-C₆ alkyl)amino, hydroxyamino, or N—C₁-C₆ alkylhydroxyamino. In some embodiments, R⁵ is R⁷—C(O)—C₁-C₆ alkyl; and R⁷ ishydroxy, C₁-C₆ alkoxy, amino, or hydroxyamino.

In some embodiments of any one of formulae (I), (Ia), and (Tb), R¹ is—NR⁵R^(5a) and R⁵ is selected from the group consisting of hydrogen,

In some embodiments of any one of formulae (I), (Ia), and (Tb), R¹ is—N(OR^(5b))R^(5a). In some embodiments, R^(5b) is hydrogen. In someembodiments, R^(5b) is C₁-C₆ alkyl. In some embodiments, R^(5b) is C₃-C₈cycloalkyl unsubstituted or substituted with one to six R⁶ and R⁶ is asdefined and described herein. In some embodiments, R^(5b) is C₃-C₈cycloalkyl unsubstituted or substituted with one to six R⁶ and each R⁶is independently hydroxy or C₁-C₆ alkyl. In some embodiments, R^(5b) isC₃-C₈ cycloalkyl-C₁-C₆ alkyl, wherein the C₃-C₈ cycloalkyl group isunsubstituted or substituted with one to six R⁶ and R⁶ is as defined anddescribed herein. In some embodiments, R^(5b) is C₃-C₈ cycloalkyl-C₁-C₆alkyl, wherein the C₃-C₈ cycloalkyl group is unsubstituted orsubstituted with one to six R⁶ and each R⁶ is independently hydroxy orC₁-C₆ alkyl. In some embodiments, R^(5b) is cyclopropyl, cyclobutyl,cyclopropyl-C₁-C₆ alkyl, or cyclobutyl-C₁-C₆ alkyl; and each of thecyclopropyl and cyclobutyl groups is unsubstituted or substituted withone to six R⁶ and R⁶ is as defined and described herein. In someembodiments, R^(5b) is cyclopropyl, cyclobutyl, cyclopropyl-C₁-C₆ alkyl,or cyclobutyl-C₁-C₆ alkyl; and each of the cyclopropyl and cyclobutylgroups is unsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl. In some embodiments, R^(5b) isC₁-C₆ hydroxyalkyl. In some embodiments, R^(5b) is C₁-C₆ alkoxy-C₁-C₆alkyl. In some embodiments, R^(5b) is amino-C₁-C₆ alkyl. In someembodiments, R^(5b) is C₁-C₆ alkylamino-C₁-C₆ alkyl. In someembodiments, R^(5b) is di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl. In someembodiments, R^(5b) is heterocycloalkyl unsubstituted or substitutedwith one to six R⁶ and R⁶ is as defined and described herein. In someembodiments, R^(5b) is heterocycloalkyl unsubstituted or substitutedwith one to six R⁶ and each R⁶ is independently hydroxy or C₁-C₆ alkyl.In some embodiments, R^(5b) is heterocycloalkyl-C₁-C₆ alkyl, wherein theheterocycloalkyl group is unsubstituted or substituted with one to sixR⁶ and R⁶ is as defined and described herein. In some embodiments,R^(5b) is heterocycloalkyl-C₁-C₆ alkyl, wherein the heterocycloalkylgroup is unsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl. In some embodiments, R^(5b) isoxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, oxetanyl-C₁-C₆ alkyl,azetidinyl-C₁-C₆ alkyl, pyrrolidinyl-C₁-C₆ alkyl, piperidinyl-C₁-C₆alkyl, or 2,2-dimethyl-1,3-dioxolan-4-yl-C₁-C₆ alkyl. In someembodiments, R^(5b) is R⁷—C(O)—C₁-C₆ alkyl; and R⁷ is hydroxy, C₁-C₆alkoxy, amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, hydroxyamino, orN—C₁-C₆ alkyl hydroxyamino. In some embodiments, R^(5b) is R⁷—C(O)—C₁-C₆alkyl; and R⁷ is hydroxy, C₁-C₆ alkoxy, amino, or hydroxyamino.

In some embodiments of any one of formulae (I), (la), and (Ib), R¹ is—N(OR^(5b))R^(5a) and —OR^(5b) is selected from the group consisting of—OH,

In some embodiments of any one of formulae (I), (la), and (Ib), R^(5a)is hydrogen. In some embodiments, R^(5a) is C₁-C₆ alkyl. In someembodiments, R^(5a) is C₁-C₄ alkyl. In some embodiments, R^(5a) ismethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, ortert-butyl. In some embodiments, R^(5a) is methyl.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R¹ is aN-linked heterocycloalkyl which is unsubstituted or substituted with oneor two R⁶, wherein R⁶ is as defined and described herein. In someembodiments, the N-linked heterocycloalkyl is N-linked azetidinyl,N-linked pyrrolidinyl, N-linked isoxazolidinyl, N-linked piperidinyl, orN-linked morpholinyl. In some embodiments, the N-linked heterocycloalkylis N-linked azetidinyl. In some embodiments, the N-linkedheterocycloalkyl is N-linked pyrrolidinyl. In some embodiments, theN-linked heterocycloalkyl is N-linked isoxazolidinyl. In someembodiments, the N-linked heterocycloalkyl is N-linked piperidinyl. Insome embodiments, the N-linked heterocycloalkyl is N-linked morpholinyl.In some embodiments, R¹ is N-linked azetidinyl which is unsubstituted orsubstituted with one or two R⁶, wherein R⁶ is as defined and describedherein. In some embodiments, R¹ is N-linked pyrrolidinyl which isunsubstituted or substituted with one or two R⁶, wherein R⁶ is asdefined and described herein. In some embodiments, R¹ is N-linkedpiperidinyl which is unsubstituted or substituted with one or two R⁶,wherein R⁶ is as defined and described herein. In some embodiments, R¹is N-linked isoxazolidinyl which is unsubstituted or substituted withone or two R⁶, wherein R⁶ is as defined and described herein. In someembodiments, R¹ is N-linked morpholinyl which is unsubstituted orsubstituted with one or two R⁶, wherein R⁶ is as defined and describedherein.

With reference to R⁶ as one or two substituents of the N-linkedheterocycloalkyl, in some embodiments, each R⁶ is independently hydroxy,oxo, or amino. In some embodiments, each R⁶ is hydroxy. In someembodiments, each R⁶ is oxo. In some embodiments, each R⁶ is amino. Insome embodiments, one of R⁶ is hydroxy and the other R⁶ is amino.

In some embodiments of any one of formulae (I), (Ia), and (Ib), R¹ is aN-linked heterocycloalkyl which is unsubstituted or substituted withhydroxy, oxo, or amino. In some embodiments, R¹ is N-linked azetidinylwhich is unsubstituted or substituted with hydroxy, oxo, or amino. Insome embodiments, R¹ is N-linked pyrrolidinyl which is unsubstituted orsubstituted with hydroxy, oxo, or amino. In some embodiments, R¹ isN-linked piperidinyl which is unsubstituted or substituted with hydroxy,oxo, or amino. In some embodiments, R¹ is N-linked isoxazolidinyl whichis unsubstituted or substituted with hydroxy, oxo, or amino. In someembodiments, R¹ is N-linked morpholinyl which is unsubstituted orsubstituted with hydroxy, oxo, or amino.

In some embodiments, the compound of formula (I) or formula (Ia) isrepresented by any one of the following formulae:

wherein R², R^(2a), R³, R^(3a), and R^(3b) are as defined herein in anyaspect or embodiment described herein.

In some embodiments of the above structures having formula (Ia), R² isiodo and R^(2a) is fluoro. In some embodiments of the above structures,R² is iodo and R^(2a) is methyl. In some embodiments of the abovestructures, R² is acetylenyl and R^(2a) is fluoro. In some embodimentsof the above structures, R² is acetylenyl and R^(2a) is methyl. In someembodiments of the above structures, R² is —SCH₃ and R^(2a) is fluoro.In some embodiments of the above structures, R² is —SCH₃ and R^(2a) ismethyl.

In some embodiments of the above structures having formula (Ia), R³,R^(3a), and R^(3b) are each hydrogen. In some embodiments of the abovestructures, R³ and R^(3a) are each hydrogen and R^(3b) is halo. In someembodiments of the above structures, R³ and R^(3a) are each hydrogen andR^(3b) is fluoro.

In some embodiments, the compound of formula (I) or formula (Ib)represented by any one of the following formulae:

wherein R², R^(2a), R³, and R^(3a) are as defined herein in any aspector embodiment described herein.

In some embodiments of the above structures having formula (Ib), R² isiodo and R^(2a) is fluoro. In some embodiments of the above structures,R² is iodo and R^(2a) is methyl. In some embodiments of the abovestructures, R² is acetylenyl and R^(2a) is fluoro. In some embodimentsof the above structures, R² is acetylenyl and R^(2a) is methyl. In someembodiments of the above structures, R² is —SCH₃ and R^(2a) is fluoro.In some embodiments of the above structures, R² is —SCH₃ and R^(2a) ismethyl.

In some embodiments of the above structures having formula (Ib), R³ andR^(3a) are each hydrogen.

Exemplified compounds of formula (I) are listed in Table 1.

TABLE 1 Compounds of formula (I) No. Structure 1.001

1.002

1.003

1.004

1.005

1.006

1.007

1.008

1.009

1.010

1.011

1.012

1.013

1.014

1.015

1.016

1.017

1.018

1.019

1.020

1.021

1.022

1.023

1.024

1.025

1.026

1.027

1.028

1.029

1.030

1.031

1.032

1.033

1.034

1.035

1.036

1.037

1.038

1.039

1.040

1.041

Additional compounds of formula (I) are listed in Table 2.

TABLE 2 Additional Compounds of formula (I) No. Structure 1.042

1.043

1.044

1.045

1.046

1.047

1.048

1.049

1.050

1.051

1.052

1.053

1.054

1.055

1.056

1.057

1.058

1.059

1.060

1.061

1.062

1.063

In some embodiments, the compound of formula (I) or (Ia) is selectedfrom the group consisting of:

In one embodiment, the compound of formula (I) or (Ib) is not ethyl6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.

Compounds in Other Forms

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Other salts include acid or base salts of the compounds used in themethods of the present invention. Illustrative examples ofpharmaceutically acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, and quaternary ammonium (methyl iodide, ethyl iodide, and thelike) salts. It is understood that the pharmaceutically acceptable saltsare non-toxic. Additional information on suitable pharmaceuticallyacceptable salts can be found in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, which isincorporated herein by reference.

Pharmaceutically acceptable salts includes salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques.

Isomers include compounds having the same number and kind of atoms, andhence the same molecular weight, but differing in respect to thestructural arrangement or configuration of the atoms.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention. Tautomerrefers to one of two or more structural isomers which exist inequilibrium and which are readily converted from one isomeric form toanother.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, the compounds of the present invention may alsocontain unnatural proportions of atomic isotopes at one or more of theatoms that constitute such compounds. For example, the compounds of thepresent invention may be labeled with radioactive or stable isotopes,such as for example deuterium (²H), tritium (³H), iodine-125 (¹²⁵I),fluorine-18 (¹⁸F), nitrogen-15 (¹⁵N), oxygen-17 (¹⁷O), oxygen-18 (¹⁸O),carbon-13 (¹³C), or carbon-14 (¹⁴C). All isotopic variations of thecompounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

IV. Composition

In another aspect, provided herein is a pharmaceutical compositionincluding the compound of formula (I) and a pharmaceutically acceptablecarrier.

The compounds provided herein can be formulated into pharmaceuticalcompositions using methods available in the art and those disclosedherein. Any of the compounds disclosed herein can be provided in theappropriate pharmaceutical composition and be administered by a suitableroute of administration.

Administration of the compound described herein to a subject may belocal or non-systemic, e.g., topical, subcutaneously, intradermal, orintralesional. In some embodiments, the compound can be administered bytopical administration. In some embodiments, the compound can beadministered by intradermal administration. In some embodiments, thecompound can be administered by intralesional administration, e.g., byintralesional injection.

The methods provided herein encompass administering pharmaceuticalcompositions containing at least one compound as described herein,including a compound of formula (I) if appropriate in a salt form,either used alone or in the form of a combination with one or morecompatible and pharmaceutically acceptable carriers, such as diluents oradjuvants, or with another agent for the treatment of a MEK-inhibitorresponsive disorder or disease, a MEK-inhibitor responsive dermaldisorder or disease, a MEK-mediated disorder or disease, or aMEK-mediated dermal disorder or disease where the subject is in needthereof.

In some embodiments, the second agent can be formulated or packaged withthe compound provided herein. Of course, the second agent will only beformulated with the compound provided herein when, according to thejudgment of those of skill in the art, such co-formulation should notinterfere with the activity of either agent or the method ofadministration. In some embodiments, the compound provided herein andthe second agent are formulated separately. They can be packagedtogether, or packaged separately, for the convenience of thepractitioner of skill in the art.

In clinical practice the active agents provided herein may beadministered by any conventional route, in particular topically,subcutaneously, intradermally, intralesionally, orally, parenterally,rectally or by inhalation (e.g. in the form of aerosols). In someembodiments, the compound provided herein is administered topically,subcutaneously, intradermally, or intralesionally. In some embodiments,the compound provided herein is administered topically. In someembodiments, the compound provided herein is administered intradermally.In some embodiments, the compound provided herein is administeredintralesionally.

Use may be made, as solid compositions for oral administration, oftablets, pills, hard gelatin capsules, powders or granules. In thesecompositions, the active product is mixed with one or more inertdiluents or adjuvants, such as sucrose, lactose or starch.

These compositions can comprise substances other than diluents, forexample a lubricant, such as magnesium stearate, or a coating intendedfor controlled release.

Use may be made, as liquid compositions for oral administration, ofsolutions which are pharmaceutically acceptable, suspensions, emulsions,syrups and elixirs containing inert diluents, such as water or liquidparaffin. These compositions can also comprise substances other thandiluents, in some embodiments, wetting, sweetening or flavoringproducts.

Use may be made, of compositions for topical administration as lotions,tinctures, creams, emulsions, gels or ointments. In these compositions,the active product is mixed with one or more inert excipients includingwater, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof.

The compositions for parenteral, intralesional, or intradermaladministration can be emulsions or sterile solutions. Use may be made,as solvent or vehicle, of propylene glycol, a polyethylene glycol,vegetable oils, in particular olive oil, or injectable organic esters,in some embodiments, ethyl oleate. These compositions can also containadjuvants, in particular wetting, isotonizing, emulsifying, dispersingand stabilizing agents. Sterilization can be carried out in severalways, in some embodiments, using a bacteriological filter, by radiationor by heating. They can also be prepared in the form of sterile solidcompositions which can be dissolved at the time of use in sterile wateror any other injectable sterile medium.

The compositions for rectal administration are suppositories or rectalcapsules which contain, in addition to the active principle, excipientssuch as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

The compositions can also be aerosols. For use in the form of liquidaerosols, the compositions can be stable sterile solutions or solidcompositions dissolved at the time of use in apyrogenic sterile water,in saline or any other pharmaceutically acceptable vehicle. For use inthe form of dry aerosols intended to be directly inhaled, the activeprinciple is finely divided and combined with a water-soluble soliddiluent or vehicle, in some embodiments, dextran, mannitol or lactose.

In some embodiments, a composition provided herein is a pharmaceuticalcomposition or a single unit dosage form. Pharmaceutical compositionsand single unit dosage forms provided herein comprise a prophylacticallyor therapeutically effective amount of one or more prophylactic ortherapeutic agents (e.g., a compound provided herein, or otherprophylactic or therapeutic agent), and a typically one or morepharmaceutically acceptable carriers or excipients. In some embodiments,the term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” includes adiluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)),excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water can be used as a carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Examples of suitable pharmaceutical carriersare described in Remington: The Science and Practice of Pharmacy;Pharmaceutical Press; 22 edition (Sep. 15, 2012).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and in some embodiments, suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions provided herein can comprise excipients thatare well known in the art and are listed, in some embodiments, in theU.S. Pharmacopeia (USP 36-NF 31 S2). In general, lactose freecompositions comprise an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose free dosage forms comprise an activeingredient, microcrystalline cellulose, pre gelatinized starch, andmagnesium stearate.

Further encompassed herein are anhydrous pharmaceutical compositions anddosage forms comprising active ingredients, since water can facilitatethe degradation of some compounds. For example, the addition of water(e.g., 5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long term storage in order to determine characteristics suchas shelf life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, New York, 1995, pp. 379 80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine can be anhydrousif substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits. Insome embodiments, suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

Further provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent, in some embodiments, in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject. The formulation shouldsuit the mode of administration. In some embodiments, the pharmaceuticalcompositions or single unit dosage forms are sterile and in suitableform for administration to a subject, in some embodiments, an animalsubject, such as a mammalian subject, in some embodiments, a humansubject.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. In some embodiments, routes ofadministration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, intramuscular, subcutaneous,oral, buccal, sublingual, inhalation, intranasal, transdermal, topical,transmucosal, intra-tumoral, intra-synovial and rectal administration.In some embodiments, the route of administration is intradermal,topical, or intralesional administration. In some embodiments, the routeof administration is non-systemic administration. In some embodiments,the composition is formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous, subcutaneous,intramuscular, oral, intranasal or topical administration to humanbeings. In some embodiments, a pharmaceutical composition is formulatedin accordance with routine procedures for subcutaneous administration tohuman beings. Typically, compositions for intravenous administration aresolutions in sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lignocamne to ease pain at the site of the injection.

In some embodiments, dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; ointments;cataplasms (poultices); pastes; powders; dressings; creams; plasters;solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to asubject, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions, oil in water emulsions, or a water in oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a subject; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a subject.

The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. In some embodiments, a dosageform used in the initial treatment of a MEK-inhibitor responsivedisorder or disease, a MEK-inhibitor responsive dermal disorder ordisease, a MEK-mediated disorder or disease, or a MEK-mediated dermaldisorder or disease may contain larger amounts of one or more of theactive ingredients it comprises than a dosage form used in themaintenance treatment of the same disorder or disease. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease or disorder. These and other ways in which specificdosage forms encompassed herein will vary from one another will bereadily apparent to those skilled in the art. See, e.g., Remington: TheScience and Practice of Pharmacy; Pharmaceutical Press; 22 edition (Sep.15, 2012).

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, in some embodiments,as a dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachet indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Typical dosage forms comprise a compound provided herein, or apharmaceutically acceptable salt, solvate or hydrate thereof lie withinthe range of from about 0.1 mg to about 1000 mg per day, given as asingle once-a-day dose in the morning or as divided doses throughout theday taken with food. Particular dosage forms can have about 0.1, 0.2,0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100,200, 250, 500 or 1000 mg of the active compound.

Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be presented as discrete dosage forms, such as, but are not limitedto, tablets (e.g., chewable tablets), caplets, capsules, and liquids(e.g., flavored syrups). Such dosage forms contain predetermined amountsof active ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington: The Scienceand Practice of Pharmacy; Pharmaceutical Press; 22 edition (Sep. 15,2012).

In some embodiments, the oral dosage forms are solid and prepared underanhydrous diseases or disorders with anhydrous ingredients, as describedin detail herein. However, the scope of the compositions provided hereinextends beyond anhydrous, solid oral dosage forms. As such, furtherforms are described herein.

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. In some embodiments, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. In some embodiments, excipientssuitable for use in solid oral dosage forms (e.g., powders, tablets,capsules, and caplets) include, but are not limited to, starches,sugars, micro crystalline cellulose, diluents, granulating agents,lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or non-aqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

In some embodiments, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

In some embodiments, excipients that can be used in oral dosage formsinclude, but are not limited to, binders, fillers, disintegrants, andlubricants. Binders suitable for use in pharmaceutical compositions anddosage forms include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208,2906, 2910), microcrystalline cellulose, and mixtures thereof.

In some embodiments, fillers suitable for use in the pharmaceuticalcompositions and dosage forms disclosed herein include, but are notlimited to, talc, calcium carbonate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre gelatinized starch, andmixtures thereof. The binder or filler in pharmaceutical compositions istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

In some embodiments, suitable forms of microcrystalline celluloseinclude, but are not limited to, the materials sold as AVICEL PH 101,AVICEL PH 103 AVICEL RC 581, AVICEL PH 105 (available from FMCCorporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.),and mixtures thereof. A specific binder is a mixture of microcrystallinecellulose and sodium carboxymethyl cellulose sold as AVICEL RC 581.Suitable anhydrous or low moisture excipients or additives includeAVICEL PH 103™ and Starch 1500 LM.

Disintegrants are used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients should be used to form solid oral dosage forms.The amount of disintegrant used varies based upon the type offormulation, and is readily discernible to those of ordinary skill inthe art. Typical pharmaceutical compositions comprise from about 0.5 toabout 15 weight percent of disintegrant, specifically from about 1 toabout 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, pre gelatinized starch, other starches, clays, otheralgins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, in some embodiments, a syloid silica gel(AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Md.), acoagulated aerosol of synthetic silica (marketed by Degussa Co. ofPlano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

Delayed Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. In some embodiments,but are not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and6,699,500; each of which is incorporated herein by reference in itsentirety. Such dosage forms can be used to provide slow or controlledrelease of one or more active ingredients using, in some embodiments,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled releaseformulations known to those of ordinary skill in the art, includingthose described herein, can be readily selected for use with the activeingredients provided herein. Thus encompassed herein are single unitdosage forms suitable for oral administration such as, but not limitedto, tablets, capsules, gel caps, and caplets that are adapted forcontrolled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the disease ordisorder in a minimum amount of time. Advantages of controlled releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased subject compliance. In addition, controlledrelease formulations can be used to affect the time of onset of actionor other characteristics, such as blood levels of the drug, and can thusaffect the occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various diseases or disorders including,but not limited to, pH, temperature, enzymes, water, or otherphysiological diseases or disorders or compounds.

In some embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In some embodiments, a pump may beused (see, Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In some embodiments, polymeric materials can be used. In someembodiments, a controlled release system can be placed in a subject atan appropriate site determined by a practitioner of skill, i.e., thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)). The active ingredient can be dispersedin a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

Parenteral Dosage Forms

In some embodiments, provided are parenteral dosage forms. In someembodiments, parenteral dosage forms can be administered to subjects byvarious routes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intra-arterial. In someembodiments, parenteral dosage forms can be administered to subjects byvarious routes including, but not limited to, topical, intradermal, orintralesional. Because their administration typically bypasses subjects'natural defenses against contaminants, parenteral dosage forms aretypically, sterile or capable of being sterilized prior toadministration to a subject. In some embodiments, parenteral dosageforms include, but are not limited to, solutions ready for injection,dry products ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. In some embodiments,suitable vehicles include, but are not limited to: Water for InjectionUSP; aqueous vehicles such as, but not limited to, Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, and Lactated Ringer's Injection; water misciblevehicles such as, but not limited to, ethyl alcohol, polyethyleneglycol, and polypropylene glycol; and non-aqueous vehicles such as, butnot limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyloleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms.

Transdermal, Topical & Mucosal Dosage Forms

Also provided are transdermal, topical, and mucosal dosage forms.Transdermal, topical, and mucosal dosage forms include, but are notlimited to, ophthalmic solutions, sprays, aerosols, creams, lotions,ointments, gels, solutions, emulsions, suspensions, or other forms knownto one of skill in the art. See, e.g., Remington: The Science andPractice of Pharmacy; Pharmaceutical Press; 22 edition (Sep. 15, 2012);and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia (1985). Dosage forms suitable for treating mucosal tissueswithin the oral cavity can be formulated as mouthwashes or as oral gels.Further, transdermal dosage forms include “reservoir type” or “matrixtype” patches, which can be applied to the skin and worn for a specificperiod of time to permit the penetration of a desired amount of activeingredients.

The term “pharmaceutically acceptable carrier” refers to apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting any subject compositionor component thereof. Each carrier must be “acceptable” in the sense ofbeing compatible with the subject composition and its components and notinjurious to the patient. Suitable carriers (e.g., excipients anddiluents) and other materials that can be used to provide transdermal,topical, and mucosal dosage forms encompassed herein are well known tothose skilled in the pharmaceutical arts, and depend on the particulartissue to which a given pharmaceutical composition or dosage form willbe applied. With that fact in mind, typical carriers include, but arenot limited to, water, acetone, ethanol, ethylene glycol, propyleneglycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate,mineral oil, and mixtures thereof to form lotions, tinctures, creams,emulsions, gels or ointments, which are nontoxic and pharmaceuticallyacceptable. In some embodiments, materials which may serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms if desired. Examples ofsuch additional ingredients are well known in the art. See, e.g.,Remington: The Science and Practice of Pharmacy; Pharmaceutical Press;22 edition (Sep. 15, 2012).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients provided. In some embodiments, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Dosage and Unit Dosage Forms

In human therapeutics, a doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, stage of the disorder ordisease and other factors specific to the subject to be treated. In someembodiments, doses are from about 1 to about 1000 mg per day for anadult, or from about 5 to about 250 mg per day or from about 10 to 50 mgper day for an adult. In some embodiments, doses are from about 5 toabout 400 mg per day or 25 to 200 mg per day per adult. In someembodiments, dose rates of from about 50 to about 500 mg per day arealso contemplated.

In further aspects, provided are methods of treating a disease ordisorder where the subject is in need thereof and/or a MEK-inhibitorresponsive disorder or disease, a MEK-inhibitor responsive dermaldisorder or disease, a MEK-mediated dermal disorder or disease, or aMEK-mediated dermal disorder or disease in a subject by administering,to a subject in need thereof, a therapeutically or prophylacticallyeffective amount of a compound provided herein, or a pharmaceuticallyacceptable salt thereof. The amount of the compound or composition whichwill be therapeutically or prophylactically effective in the treatmentof a disorder or one or more symptoms thereof will vary with the natureand severity of the disease or condition, and the route by which theactive ingredient is administered. The frequency and dosage will alsovary according to factors specific for each subject depending on thespecific therapy (e.g., therapeutic or prophylactic agents)administered, the severity of the disorder, disease, or condition, theroute of administration, as well as age, body, weight, response, and thepast medical history of the subject. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro or animal model testsystems.

In some embodiments, exemplary doses of a composition include milligramor microgram amounts of the active compound per kilogram of subject orsample weight (e.g., about 10 micrograms per kilogram to about 50milligrams per kilogram, about 100 micrograms per kilogram to about 25milligrams per kilogram, or about 100 microgram per kilogram to about 10milligrams per kilogram). For compositions provided herein, in someembodiments, the dosage administered to a subject is 0.140 mg/kg to 3mg/kg of the subject's body weight, based on weight of the activecompound. In some embodiments, the dosage administered to a subject isbetween 0.20 mg/kg and 2.00 mg/kg, between 0.30 mg/kg and 1.50 mg/kg,between 1 mg/kg and 100 mg/kg, between 5 mg/kg and 50 mg/kg, between 10mg/kg and 50 mg/kg, between 20 mg/kg and 50 mg/kg, between 15 mg/kg and40 mg/kg, between 15 mg/kg and 35 mg/kg, between 15 mg/kg and 30 mg/kg,between 25 mg/kg and 35 mg/kg, between 10 mg/kg and 30 mg/kg, between 10mg/kg and 20 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40mg/kg, about 45 mg/kg, or about 50 mg/kg of the subject's body weight.

In some embodiments, the recommended daily dose range of a compositionprovided herein for the diseases or disorders described herein liewithin the range of from about 0.1 mg to about 1000 mg per day, given asa single once-a-day dose or as divided doses throughout a day. In someembodiments, the daily dose is administered twice daily in equallydivided doses. In some embodiments, a daily dose range should be fromabout 10 mg to about 200 mg per day, in some embodiments, between about10 mg and about 150 mg per day, in further embodiments, between about 25and about 100 mg per day. It may be necessary to use dosages of theactive ingredient outside the ranges disclosed herein in some cases, aswill be apparent to those of ordinary skill in the art. Furthermore, itis noted that the clinician or treating physician will know how and whento interrupt, adjust, or terminate therapy in conjunction with subjectresponse.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the compositionprovided herein are also encompassed by the herein described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. In some embodiments, the dosageadministered to the subject may be increased to improve the prophylacticor therapeutic effect of the composition or it may be decreased toreduce one or more side effects that a particular subject isexperiencing.

In some embodiments, the dosage of the composition provided herein,based on weight of the active compound, administered to prevent, treat,manage, or ameliorate a disorder, or one or more symptoms thereof in asubject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. In someembodiments, the dosage of the composition or a composition providedherein administered to prevent, treat, manage, or ameliorate a disorder,or one or more symptoms thereof in a subject is a unit dose of 0.1 mg to200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg,0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to5 mg, or 1 mg to 2.5 mg.

In some embodiments, treatment or prevention can be initiated with oneor more loading doses of a compound or composition provided hereinfollowed by one or more maintenance doses. In such embodiments, theloading dose can be, for instance, about 60 to about 400 mg per day, orabout 100 to about 200 mg per day for one day to five weeks. The loadingdose can be followed by one or more maintenance doses. In someembodiments, each maintenance does is, independently, about from about10 mg to about 200 mg per day, between about 25 mg and about 150 mg perday, or between about 25 and about 80 mg per day. Maintenance doses canbe administered daily and can be administered as single doses, or asdivided doses.

In some embodiments, a dose of a compound or composition provided hereincan be administered to achieve a steady-state concentration of theactive ingredient in blood or serum of the subject. The steady-stateconcentration can be determined by measurement according to techniquesavailable to those of skill or can be based on the physicalcharacteristics of the subject such as height, weight and age. In someembodiments, a sufficient amount of a compound or composition providedherein is administered to achieve a steady-state concentration in bloodor serum of the subject of from about 300 to about 4000 ng/mL, fromabout 400 to about 1600 ng/mL, or from about 600 to about 1200 ng/mL. Insome embodiments, loading doses can be administered to achievesteady-state blood or serum concentrations of about 1200 to about 8000ng/mL, or about 2000 to about 4000 ng/mL for one to five days. In someembodiments, maintenance doses can be administered to achieve asteady-state concentration in blood or serum of the subject of fromabout 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, orfrom about 600 to about 1200 ng/mL.

In some embodiments, administration of the same composition may berepeated and the administrations may be separated by at least 1 day, 2days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or 6 months. In some embodiments, administration of thesame prophylactic or therapeutic agent may be repeated and theadministration may be separated by at least at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months.

In certain aspects, provided herein are unit dosages comprising acompound, or a pharmaceutically acceptable salt thereof, in a formsuitable for administration. Such forms are described in detail herein.In some embodiments, the unit dosage comprises 1 to 1000 mg, 5 to 250 mgor 10 to 50 mg active ingredient. In particular embodiments, the unitdosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mgactive ingredient. Such unit dosages can be prepared according totechniques familiar to those of skill in the art.

The dosage may vary within a range depending upon the dosage formemployed and the route of administration utilized. For any compound, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve alevel in the skin with the lesion, e.g., the dermal neurofibroma, thesubdermal neurofibroma, or the superficial plexiform neurofibroma) thatincludes the IC₅₀ (i.e., the concentration of the test compound thatachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. In addition, levels in plasma may be measured,for example, by high performance liquid chromatography, in order toascertain systemic exposure.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, the size of the lesion, number of lesions, general health, sex,diet, time of administration, drug combination, and the judgment of thetreating physician and the severity of the particular disease beingtreated. The amount of a soft MEK inhibitor, e.g., a soft MEK inhibitordescribed herein, in the composition will also depend upon theparticular soft MEK inhibitor in the composition.

In some embodiments, the topical dose is about 0.01 μg/cm², about 0.05μg/cm², about 0.1 μg/cm², about 0.15 μg/cm², about 0.2 μg/cm², about 0.3μg/cm², about 0.4 μg/cm², about 0.5 μg/cm², about 0.6 μg/cm², about 0.7μg/cm², about 0.8 μg/cm², or about 0.9 μg/cm²; or is within about0.01-0.03 μg/cm², about 0.03-0.05 μg/cm², about 0.05-0.1 μg/cm², about0.1-0.3 μg/cm², about 0.3-0.5 μg/cm², about 0.5-0.8 μg/cm², about0.8-1.0 μg/cm², about 1-10 μg/cm², about 10-20 μg/cm², about 20-30μg/cm², about 30-40 μg/cm², about 40-50 μg/cm², about 50-60 μg/cm²,about 60-70 μg/cm², about 70-80 μg/cm², about 80-90 μg/cm², about 90-100μg/cm², about 100-125 μg/cm², about 125-150 sg/cm², about 150-175μg/cm², about 175-200 μg/cm², about 200-250 μg/cm², about 250-300μg/cm², about 300-350 μg/cm², about 350-400 μg/cm², about 400-450μg/cm², about 450-500 μg/cm², about 500-550 μg/cm², about 550-600μg/cm², about 600-650 μg/cm², about 650-700 μg/cm², about 700-750μg/cm², about 750-800 μg/cm², about 800-850 μg/cm², about 850-900μg/cm², about 900-950 μg/cm², or about 950-1000 μg/cm².

In some embodiments, the topical dose is within about 0.5-1.0 mg/cm²,1.0-1.5 mg/cm², 1.5-2.0 mg/cm², 2.5-2.5 mg/cm², 3.0-3.5 mg/cm², 3.5-5.0mg/cm², 5.0-7.5 mg/cm², 7.5-10 mg/cm², 1-10 mg/cm², about 10-20 mg/cm²,about 20-30 mg/cm², about 30-40 mg/cm², about 40-50 mg/cm², about 50-60mg/cm², about 60-70 mg/cm², about 70-80 mg/cm², about 80-90 mg/cm²,about 90-100 mg/cm², about 100-125 mg/cm², about 125-150 mg/cm², about150-175 mg/cm², about 175-200 mg/cm², about 200-250 mg/cm², about250-300 mg/cm², about 300-350 mg/cm², about 350-400 mg/cm², about400-450 mg/cm², about 450-500 mg/cm², about 500-550 mg/cm², about550-600 mg/cm², about 600-650 mg/cm², about 650-700 mg/cm², about700-750 mg/cm², about 750-800 mg/cm², about 800-850 mg/cm², about850-900 mg/cm², about 900-950 mg/cm², or about 950-1000 mg/cm².

V. Kits

Also provided are kits for use in methods of treatment of aMEK-inhibitor responsive disorder or disease, a MEK-inhibitor responsivedermal disorder or disease, a MEK-mediated disorder or disease, or aMEK-mediated dermal disorder or disease where the subject is in needthereof; or a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease. Thekits can include a compound or composition provided herein, a secondagent or composition, and instructions providing information to a healthcare provider regarding usage for treating a MEK-inhibitor responsivedisorder or disease, a MEK-inhibitor responsive dermal disorder ordisease, a MEK-mediated disorder or disease, or a MEK-mediated dermaldisorder or disease. Instructions may be provided in printed form or inthe form of an electronic medium such as a floppy disc, CD, or DVD, orin the form of a website address where such instructions may beobtained. A unit dose of a compound or composition provided herein, or asecond agent or composition, can include a dosage such that whenadministered to a subject, a therapeutically or prophylacticallyeffective plasma level of the compound or composition can be maintainedin the subject for at least 1 day. In some embodiments, a compound orcomposition can be included as a sterile aqueous pharmaceuticalcomposition or dry powder (e.g., lyophilized) composition.

In some embodiments, suitable packaging is provided. As used herein,“packaging” includes a solid matrix or material customarily used in asystem and capable of holding within fixed limits a compound providedherein and/or a second agent suitable for administration to a subject.Such materials include glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes and the like. If e-beam sterilizationtechniques are employed, the packaging should have sufficiently lowdensity to permit sterilization of the contents.

VI. Methods

In a third aspect, provided herein is a method for treating a disease ordisorder where the subject is in need thereof and the disease ordisorder is a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease in asubject. The method includes administering the subject with atherapeutically or prophylactically effective amount of a compounddisclosed herein, e.g., a compound of formula (I), and compounds inTables 1 and 2, including a single enantiomer, a mixture of anenantiomeric pair, an individual diastereomer, a mixture ofdiastereomers, an individual stereoisomer, a mixture of stereoisomers,or a tautomeric form thereof; or a pharmaceutically acceptable salt,solvate, prodrug, phosphate, or active metabolite thereof.

In some embodiments, the method includes administering the subject witha therapeutically effective amount of a compound of formula (I) andcompounds in Tables 1 and 2, including a single enantiomer, a mixture ofan enantiomeric pair, an individual diastereomer, a mixture ofdiastereomers, an individual stereoisomer, a mixture of stereoisomers,or a tautomeric form thereof; or a pharmaceutically acceptable salt orsolvate thereof. In some embodiments, the method includes administeringthe subject with a therapeutically effective amount of a compound offormula (I) and compounds in Table 1, including a single enantiomer, amixture of an enantiomeric pair, an individual diastereomer, a mixtureof diastereomers, an individual stereoisomer, a mixture ofstereoisomers, or a tautomeric form thereof; or a pharmaceuticallyacceptable salt or solvate thereof.

In some embodiments, the MEK-inhibitor responsive dermal disorder orMEK-mediated dermal disorder is selected from the group consisting ofdermal rasopathy, neurofibromatosis type 1, dermal neurofibroma,subdermal neurofibroma, and superficial plexiform neurofibroma.

In some embodiments, the MEK-inhibitor responsive dermal disorder orMEK-mediated dermal disorder is neurofibromatosis type 1.

In some embodiments, administering includes contacting the soft MEKinhibitor with the skin, mucous membranes, vagina, penis, larynx, vulva,cervix, or anus of the subject, by local or non-systemic application,e.g., topical, intradermal, or intralesional application or applicationby suppository, of the soft MEK inhibitor.

In some embodiments, the tumor associated with neurofibromatosis type 1(NF1), e.g., a dermal neurofibroma, a subdermal neurofibroma, or asuperficial plexiform neurofibroma, is reduced, e.g., the size or thetotal tumor volume is reduced, by at least about 15% relative to thereference standard (e.g., from about 15% to about 60%), thereby treatingthe subject. In some embodiments, the reference standard is the size orthe total tumor volume in an untreated control, e.g., from the samesubject or a different subject.

In some embodiments, the size or total tumor volume of the tumorassociated with neurofibromatosis type 1 (NF1), e.g., a dermalneurofibroma, a subdermal neurofibroma, or a superficial plexiformneurofibroma, is reduced by at least about 15%, by at least about 20%,by at least about 25%, by at least about 30%, by at least about 35%, byat least about 40%, by at least about 45%, by at least about 50%, by atleast about 55%, by at least about 60% relative to the referencestandard. In some embodiments, the reference standard is the size or thetotal tumor volume in an untreated control, e.g., from the same subjector a different subject.

In some embodiments, the method includes evaluating the subject withmagnetic resonance imaging (MRI), or optical imaging, e.g., evaluatingthe volume of tumors obtained from the subject, e.g., prior to, duringand/or after treatment.

Neurofibromatosis type 1 (NF1): In some embodiments, the dermal disorderis associated with NF1. NF1, also known as von RecklinghausenNeurofibromatosis or Peripheral Neurofibromatosis, occurs inapproximately 1:3,000 births, and is one of the most prevalent geneticdisorders and the most common neurocutaneous disorders. NF1 is caused bya deficiency in neurofibromin, which leads to hyperactivation of variouscell-signaling pathways, e.g., Ras and Rho, is associated with severaldermal disorders, including dermal neurofibromas (DFs); subdermalneurofibromas; superficial plexiform neurofibromas (PFs); cutaneousneurofibromas (CFs); café au lait spots; and axillary and inguinalfreckling. DFs occur in over 95% of NF1 patients. DFs can appearanywhere on the body, with 88% of NF1 patients over 40 years of agehaving over 100 DFs. DFs can cause both severe physical pain,disfigurement, as well as social anxiety. Facial DFs can createsignificant social anxiety issues and pain among affected individuals.DFs (also known as cutaneous neurofibromas or discrete neurofibromas)grow from small nerves in the skin or just under the skin and appear assmall bumps typically beginning around the time of puberty. Currenttreatment options for DF are limited to surgical excisin and CO₂ laserremoval, both of which cause scarring and neither of which ispreventative.

Other Dermal Rasopathies: In some embodiments, the dermal disorder isassociated with enhanced activation of Ras. In some embodiments, thedermal disorder is selected from: psoriasis, keratocanthoma (KA),hyperkeratosis, papilloma, Noonan syndrome (NS), cardiofaciocutaneoussyndrome (CFC), Costello syndrome (faciocutaneoskeletal syndrome or FCSsyndrome), oculoectodermal syndrome, cafe au lait spots and Multiplelentigines syndrome (formerly called Leopard syndrome).

In some or any embodiments, the disease to be reduced, ameliorated,treated, or prevented is not cancer (e.g. melanoma).

In some embodiments, the disease to be reduced, ameliorated, treated, orprevented is cancer, a dermal rasopathy, a dermal disorder associatedwith neurofibromatosis type 1, a dermal neurofibroma, a subdermalneurofibroma, or a superficial plexiform neurofibroma, psoriasis,keratocanthoma (KA), hyperkeratosis, papilloma, Noonan syndrome (NS),cardiofaciocutaneous syndrome (CFC), Costello syndrome(faciocutaneoskeletal syndrome or FCS syndrome), oculoectodermalsyndrome, cafe au lait spots, and Multiple lentigines syndrome (formerlycalled Leopard syndrome).

In some embodiments, the disease to be reduced, ameliorated, treated, orprevented is cancer. In some embodiments, the disease to be reduced,ameliorated, treated, or prevented is selected from the group consistingof basal cell carcinoma, squamous cell carcinoma, aktinic keratosis,Kaposi's sarcoma, dermal lymphoma, cervical cancer, HPV-related squamouscell carcinoma, and melanoma.

In some embodiments, the disease to be reduced, ameliorated, treated, orprevented is a dermal rasopathy, a dermal disorder associated withneurofibromatosis type 1, a dermal neurofibroma, a subdermalneurofibroma, or a superficial plexiform neurofibroma, psoriasis,keratocanthoma (KA), hyperkeratosis, papilloma, Noonan syndrome (NS),cardiofaciocutaneous syndrome (CFC), Costello syndrome(faciocutaneoskeletal syndrome or FCS syndrome), oculoectodermalsyndrome, cafe au lait spots, and Multiple lentigines syndrome (formerlycalled Leopard syndrome).

In some embodiments, the compounds described herein are used for thereduction of a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease, wherethe subject is in need thereof.

In some embodiments, the compounds described herein are used for theamelioration of a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease, wherethe subject is in need thereof.

In some embodiments, the compounds described herein are used forprevention of a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease, wherethe subject is in need thereof.

In some embodiments, the compounds described herein are used fortreatment of a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease, wherethe subject is in need thereof.

Assay Methods

Compounds can be assayed for efficacy in treating a MEK-inhibitorresponsive disorder or disease, a MEK-inhibitor responsive dermaldisorder or disease, a MEK-mediated disorder or disease, or aMEK-mediated dermal disorder or disease where the subject is in needthereof according to any assay known to those of skill in the art.Exemplary assay methods are provided elsewhere herein.

VII. Combination Therapies

In some embodiments, the compounds and compositions provided herein areuseful in methods of treatment of a MEK-inhibitor responsive disorder ordisease, a MEK-inhibitor responsive dermal disorder or disease, aMEK-mediated disorder or disease, or a MEK-mediated dermal disorder ordisease where the subject is in need thereof, that comprise furtheradministration of a second agent effective for the treatment of dermaldisorders or diseases. The second agent can be any agent known to thoseof skill in the art to be effective for the treatment of dermaldisorders or diseases, including those currently approved by the UnitedStates Food and Drug Administration, or other similar body of a countryforeign to the United States.

In some embodiments, a compound provided herein is administered incombination with one second agent. In further embodiments, a compoundprovided herein is administered in combination with two second agents.In still further embodiments, a compound provided herein is administeredin combination with two or more second agents.

In some embodiments, the methods encompass the step of administering tothe subject in need thereof an amount of a compound effective for thetreatment of a MEK-inhibitor responsive disorder or disease, aMEK-inhibitor responsive dermal disorder or disease, a MEK-mediateddisorder or disease, or a MEK-mediated dermal disorder or disease wherethe subject is in need thereof in combination with a second agenteffective for the treatment or prevention of a MEK-inhibitor responsivedisorder or disease, a MEK-inhibitor responsive dermal disorder ordisease, a MEK-mediated disorder or disease, or a MEK-mediated dermaldisorder or disease where the subject is in need thereof. The compoundcan be any compound as described herein, and the second agent can be anysecond agent described in the art or herein. In some embodiments, thecompound is in the form of a pharmaceutical composition or dosage form,as described elsewhere herein.

As used herein, the term “in combination” includes the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).The use of the term “in combination” does not restrict the order inwhich therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a disorder. A first therapy (e.g., aprophylactic or therapeutic agent such as a compound provided herein)can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disorder.

As used herein, the term “synergistic” includes a combination of acompound provided herein and another therapy (e.g., a prophylactic ortherapeutic agent) which has been or is currently being used to prevent,manage or treat a disorder, which is more effective than the additiveeffects of the therapies. A synergistic effect of a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents)permits the use of lower dosages of one or more of the therapies and/orless frequent administration of said therapies to a subject with adisorder. The ability to utilize lower dosages of a therapy (e.g., aprophylactic or therapeutic agent) and/or to administer said therapyless frequently reduces the toxicity associated with the administrationof said therapy to a subject without reducing the efficacy of saidtherapy in the prevention or treatment of a disorder). In addition, asynergistic effect can result in improved efficacy of agents in theprevention or treatment of a disorder. Finally, a synergistic effect ofa combination of therapies (e.g., a combination of prophylactic ortherapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of either therapy alone.

The active compounds provided herein can be administered in combinationor alternation with another therapeutic agent, in particular an agenteffective in the treatment of a MEK-inhibitor responsive disorder ordisease, a MEK-inhibitor responsive dermal disorder or disease, aMEK-mediated disorder or disease, or a MEK-mediated dermal disorder ordisease where the subject is in need thereof. In combination therapy,effective dosages of two or more agents are administered together,whereas in alternation or sequential-step therapy, an effective dosageof each agent is administered serially or sequentially. The dosagesgiven will depend on absorption, inactivation and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of theMEK-inhibitor responsive disorder or disease, the MEK-inhibitorresponsive dermal disorder or disease, the MEK-mediated disorder ordisease, or the MEK-mediated dermal disorder or disease or a disorder tobe alleviated. It is to be further understood that for any particularsubject, specific dosage regimens and schedules should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions.

In some embodiments, dosages of the second agents to be used in acombination therapy are provided herein. In some embodiments, dosageslower than those which have been or are currently being used to treat aMEK-inhibitor responsive disorder or disease, a MEK-inhibitor responsivedermal disorder or disease, a MEK-mediated disorder or disease, or aMEK-mediated dermal disorder or disease are used in the combinationtherapies provided herein. The recommended dosages of second agents canbe obtained from the knowledge of those of skill in the art. For thosesecond agents that are approved for clinical use, recommended dosagesare described in, for example, Hardman et al., eds., 1996, Goodman &Gilman's The Pharmacological Basis Of Therapeutics 9^(th) Ed,Mc-Graw-Hill, New York; Physician's Desk Reference (PDR) 57^(th) Ed.,2003, Medical Economics Co., Inc., Montvale, N.J.; which areincorporated herein by reference in their entirety.

The disclosure provides combination treatments by administration of asoft MEK inhibitor described herein with one or more additionalagent(s). In some embodiments, the one or more additional agent(s) isselected from:

-   -   agents that treat acne (e.g., Accutane, Azelaic acid, Benzoil        Peroxide, Salicylic acid);    -   analgesics (e.g., Acetaminophen, Capsaicin), e.g., a Cox2        Inhibitor, e.g. Celecoxib);    -   anesthetics (e.g., Benzocaine, Benzocaine/Menthol, Dibucaine,        Diperodon, Lidocaine, Lidocaine/Prilocaine, Pramoxine);    -   anti-infectives (e.g., Crotamiton);    -   anti-prurittus (e.g., Ammonium lactate, Benzocaine, an ascomycin        macrolactam, e.g., Pimecrolimus);    -   anti-prurittus/5HT3 receptor antagonists (e.g., Ondansetron);    -   antibiotics (e.g., clindamycin, doxycycline, erythromycin,        tetracycline);    -   anticholinergic antiemetics (e.g., diphenhydramine);    -   antifibrotics (e.g., Collagenase, Pirfenidone);    -   antihistamines (e.g., Triprolidine (Actifed@), Fexofenadine        (Allergra®, Allegra® D-12, Allegra®-24), Astepro/Astelin Nasal        Spray (Azalastine) (Dymista®), Hydroxyzine hydrochloride        (Atarax®), Diphenhydramine Hydrochloride (Benadryl®),        Brompheniramine (Dimetapp® Cold and Allergy Elixir), Zyrtec®        (Cetirizine), Chlor-Trimeton® (Chlorpheniramine), Descoratadine        (Clarinex®, Clarinex® D-12, and Clarinex® D-24), Loratadine        (Claritin®, Claritin® D-12, Claritin® D-24, and Alavert®),        Dimenhydrinate (Dramamine®), Diphenhydramine (Benadryl® Allergy,        Nytol®, Sominex®), Doxylamine (Vicks® NyQuil®, Alka-Seltzer®        Plus Night-Time Cold Medicine), Cyproheptadine (Periactin®),        Promethazine (Phenergan®), Acrivastine (Semprex®, Semprex®-D),        Clemastine (Tavist®), doxylamine (Unisom®), Levoceterizine        (Xyzal®);    -   mast cell stabalizers (e.g. Beta2-adrenergic agonists,        Cromoglicic acid, cromolyn sodium, Gastrocrom®, Ketotifen,        Methylxanthines, Omalizumab, Pemirolast, Quercetin, Ketotifen        (Zaditen®));    -   anti-inflammatory agents (e.g., NSAID (e.g. Aspirin, Choline and        magnesium salicylates, Diclofenac potassium (Cataflam®),        Diclofenac sodium (Voltaren®, Voltaren® XR), Diclofenac sodium        with misoprostol (Arthrotec®), Diflunisal (Dolobid®), Etodolac        (Lodine®, Lodine® XL), Fenoprofen calcium (Nalfon®),        Flurbiprofen (Ansaid®), ibuprofen (Advil®, Motrin®, Motrin® IB,        Nuprin®), Indomethacin (Indocin®, Indocin® SR), Ketoprofen        (Actron®, Orudis®, Orudis® KT, Oruvail®), Magnesium salicylate        (Arthritab, Bayer® Select, Doan's Pills, Magan, Mobidin,        Mobogesic) Meclofenamate sodium (Meclomen®), Mefenamic acid        (Ponstel®), Meloxicam (Mobic®), Nabumetone (Relafen®), Naproxen        (Naprosyn®, Naprelan®), Naproxen sodium (Aleve®, Anaprox®),        Oxaprozin (Daypro®), Piroxicam (Feldene®), Rofecoxib (Vioxx®),        Salsalate (Amigesic, Anaflex 750, Disalcid, Marthritic,        Mono-Gesic, Salflex, Salsitab), Sodium salicylate, Sulindac        (Clinoril®), Tolmetin sodium (Tolectin®), Valdecoxib (Bextra®));    -   Receptor Tyrosine Kinase Inhibitor (e.g. Sunitinib);    -   Alkylating Agents (e.g., Dacarbazine, Carboplatin);    -   CDK 4/6 Inhibitors (e.g., LEE011);    -   PKC Inhibitors (e.g., AEB071);    -   MAPK inhibitors (e.g., RAS Inhibitors/Farnesyltransferase        inhibitor (e.g. Tipifamib), Raf Kinase Inhibitor (e.g. Sorafenib        (BAY 43-9006, Nexavar), Vemurafenib, Dabrafenib, LGX818,        TAK-632, MLN2480, PLX-4720), ERK Inhibitors (e.g., SCH772984,        VTX11e);    -   P13K Inhibitor (e.g., LY294002);    -   AKT Inhibitor (e.g., MK 2206);    -   P13K/AKT Inhibitor (e.g. buparlisib, Cixutumumab);    -   mTOR Inhibitors (e.g. Topical Rapamycin, RAD001        (Everolimus/Rapamycin), Temsirolimus, Sirolimus);    -   Tyrosine Kinase Inhibitors (e.g. Imatinib (Gleevec®),        Cabozantinib (inhibitor of tyrosine kinases c-Met and VEGFR2),        Nilotinib (Tasigna®);    -   VEGF Inhibitor (e.g. Ranibizumab (Lucentis@), Cediranib);    -   Immune Response Modifier (e.g. Topical Imiquimod, Interferon,        PEG Interferon);    -   Calcium Channel Blocker (e.g. Avocil (Mederma)/15% Verapamil,        vitamin D separately, Doxycyline Injections);    -   Statin (e.g. Lovastatin, Methotrexate, Vinblastine, Pregabalin,        Temozolomide, PLX3397);    -   HDAC Inhibitor (e.g. AR-42);    -   HSP-90 Inhibitors (e.g. Ganetespib);    -   retinoids (e.g. adapalene, Isotretinoin, tazarotene, tretinoin);    -   steroids (e.g. Alclometasone, Amcinonide, Betamethasone,        Betamethasone dipropionate, Betamethasone dipropionate,        augmented, Budesonide, Clobetasol propionate, Cortisone,        Desonide, Dexamethasone, Diflorasone diacetate, Fluocinolone        acetonide, Fluocinonide, Flurandrenolide, Fluticasone        propionate, Halobetasol propionate, Halocinonide,        Hydrocortisone, Hydrocortisone butyrate, Hydrocortisone        valerate, Methylprednisolone, Mometasone, Mometasone furoate,        Prednicarbate, Prednisolone, Prednisone, Triamcinolone,        Triamcinolone acetonide);    -   topical calcineurin inhibitors (e.g., pimecrolimus (Elidel®        Cream 1%, Novartis, tacrolimus (Protopic® Ointment, Astellas));        and    -   Non-pharmaceutical Interventions (e.g. photodynamic Therapy        (Levulan Kerastick Topical+light), Electrodesication (ED), YAG        Laser).

In various embodiments, the therapies (e.g., a compound provided hereinand the second agent) are administered less than 5 minutes apart, lessthan 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hoursapart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hoursto 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hoursapart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96hours to 120 hours apart. In various embodiments, the therapies areadministered no more than 24 hours apart or no more than 48 hours apart.In some embodiments, two or more therapies are administered within thesame patient visit. In some embodiments, the compound provided hereinand the second agent are administered concurrently.

In some embodiments, the compound provided herein and the second agentare administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In some embodiments, administration of the same agent may be repeatedand the administrations may be separated by at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In some embodiments, administration of the sameagent may be repeated and the administration may be separated by atleast at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days,45 days, 2 months, 75 days, 3 months, or 6 months.

In some embodiments, a compound provided herein and a second agent areadministered to a patient, in some embodiments, a mammal, such as ahuman, in a sequence and within a time interval such that the compoundprovided herein can act together with the other agent to provide anincreased benefit than if they were administered otherwise. In someembodiments, the second active agent can be administered at the sametime or sequentially in any order at different points in time; however,if not administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeutic orprophylactic effect. In some embodiments, the compound provided hereinand the second active agent exert their effect at times which overlap.Each second active agent can be administered separately, in anyappropriate form and by any suitable route. In some embodiments, thecompound provided herein is administered before, concurrently or afteradministration of the second active agent.

In some embodiments, the compound provided herein and the second agentare cyclically administered to a patient. Cycling therapy involves theadministration of a first agent (e.g., a first prophylactic ortherapeutic agent) for a period of time, followed by the administrationof a second agent and/or third agent (e.g., a second and/or thirdprophylactic or therapeutic agent) for a period of time and repeatingthis sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improve theefficacy of the treatment.

In some embodiments, the compound provided herein and the second activeagent are administered in a cycle of less than about 3 weeks, about onceevery two weeks, about once every 10 days or about once every week. Onecycle can comprise the administration of a compound provided herein andthe second agent by infusion over about 90 minutes every cycle, about 1hour every cycle, about 45 minutes every cycle. Each cycle can compriseat least 1 week of rest, at least 2 weeks of rest, at least 3 weeks ofrest. The number of cycles administered is from about 1 to about 12cycles, more typically from about 2 to about 10 cycles, and moretypically from about 2 to about 8 cycles.

In some embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecompound provided herein can work together with the second active agent.In some embodiments, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day.

The second agent can act additively or synergistically with the compoundprovided herein. In some embodiments, the compound provided herein isadministered concurrently with one or more second agents in the samepharmaceutical composition. In some embodiments, a compound providedherein is administered concurrently with one or more second agents inseparate pharmaceutical compositions. In some embodiments, a compoundprovided herein is administered prior to or subsequent to administrationof a second agent. Also contemplated are administration of a compoundprovided herein and a second agent by the same or different routes ofadministration, e.g., oral and parenteral. In some embodiments, when thecompound provided herein is administered concurrently with a secondagent that potentially produces adverse side effects including, but notlimited to, toxicity, the second active agent can advantageously beadministered at a dose that falls below the threshold that the adverseside effect is elicited.

VIII. Examples

General Synthetic Methods

The compounds provided herein can be prepared, isolated or obtained byany method apparent to those of skill in the art. Compounds providedherein can be prepared according to the Exemplary Preparation Schemesprovided below. Reaction conditions, steps and reactants not provided inthe Exemplary Preparation Schemes would be apparent to, and known by,those skilled in the art. As used herein, the symbols and conventionsused in these processes, schemes and examples, regardless of whether aparticular abbreviation is specifically defined, are consistent withthose used in the contemporary scientific literature, for example, theJournal of the American Chemical Society or the Journal of BiologicalChemistry. Specifically, but without limitation, the followingabbreviations may be used in the examples and throughout thespecification: g (grams); mg (milligrams); mL (milliliters); μL(microliters); mM (millimolar); μM (micromolar); Hz (Hertz); MHz(megahertz); mmol (millimoles); hr or hrs (hours); min (minutes); MS(mass spectrometry); ESI (electrospray ionization); TLC (thin layerchromatography); HPLC (high pressure liquid chromatography); THF(tetrahydrofuran); CDCl₃ (deuterated chloroform); AcOH (acetic acid);DCM (dichloromethane); DMSO (dimethylsulfoxide); DMSO-d₆ (deuterateddimethylsulfoxide); EtOAc (ethyl acetate); MeOH (methanol); Tces(2,2,2-trichloroethoxysulfonyl); —Si(tert-Bu)(Ph)₂ and —Si^(t) BuPh₂(tert-butyl-diphenylsilyl); and BOC (t-butyloxycarbonyl).

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCelsius). All reactions are conducted at room temperature unlessotherwise noted. Synthetic methodologies illustrated herein are intendedto exemplify the applicable chemistry through the use of specificexamples and are not indicative of the scope of the disclosure.

Compounds of formula (Ia) can be prepared according to Scheme 1 as shownin FIG. 1 , in which R¹ is —OR⁴, —NR⁵R^(5a), —N(OR^(5b))R^(5a), or aN-linked heterocycloalkyl which is unsubstituted or substituted with oneor two R⁶; and X, R², R^(2a), R³, R^(3a), R⁴, R⁵, R^(5a), R^(5b), and R⁶are as defined in any aspect, embodiment, or claim as described herein.In some embodiments, R³ and R^(3a) are each hydrogen.

Starting from commercially-available or routinely-accessible acids offormula (101) and commercially-available or routinely-accessibleanilines of formula (102), compounds of formula (103) can be prepared bymethods apparent to those of skill in the art. The acid-containingcompound of formula (103) can be activated with numerous reagentsapparent to those of skill in the art to produce compounds with asuitable leaving group attached to the carbonyl of the C(O)OH acidgroup, for example an acid chloride produced from the reaction of (103)with thionyl chloride or an active ester produced from the reaction of(103) with reagents such as EDCI or HOBt. The acid chlorides or activeesters can then be reacted with compounds of formula R⁴OH, HNR⁵R^(5a),HN(OR^(5b))R^(5a), R^(a)—H (where R^(a) is a N-linked heterocycloalkylunsubstituted or substituted with one or two R⁶), or suitable protectedforms thereof to produce compounds of formula (Ia).

Compounds of formula (Ia) can be prepared according to Scheme 2 as shownin FIG. 2 , in which R¹ is —OR⁴, —NR⁵R^(5a), —N(OR^(5b))R^(5a), or aN-linked heterocycloalkyl unsubstituted or substituted with one or twoR⁶; R² is iodo; R^(3b) is fluoro; and R^(2a), R³, R^(3a), R⁴, R⁵,R^(5a), R^(5b), and R⁶ are as defined in any aspect, embodiment, orclaim as described herein. In some embodiments, R³ and R^(3a) are eachhydrogen.

Starting from commercially-available or routinely-accessible acids offormula (104) and commercially-available or routinely-accessibleanilines of formula (105), compounds of formula (106) can be prepared bymethods apparent to those of skill in the art. A compound of formula(106) is then treated with Zn(CN)₂ in the presence of a catalyst such asPd(PPh₃)₄. The compound of formula (108) is prepared by treating (107)with iodine in the presence of siver trifluoroacetate or alternativelywith iodine monochloride. The acid-containing compound of formula (108)can be activated with numerous reagents apparent to those of skill inthe art to produce compounds with a suitable leaving group attached tothe carbonyl of the C(O)OH acid group, for example an acid chlorideproduced from the reaction of (108) with thionyl chloride or an activeester produced from the reaction of (108) with reagents such as EDCI orHOBt. The acid chlorides or active esters can then be reacted withcompounds of formula R⁴OH, HNR⁵R^(5a), HN(OR^(5b))R^(5a), R^(a)—H (whereR^(a) is a N-linked heterocycloalkyl unsubstituted or substituted withone or two R⁶), or suitable protected forms thereof to produce compoundsof formula (Ia), in which R² is iodo and R^(3b) is fluoro.

Compounds of formula (Ib) can be prepared according to Scheme 3 as shownin FIG. 3 , in which R¹ is —OR⁴, —NR⁵R^(5a), —N(OR^(5b))R^(5a), or aN-linked heterocycloalkyl unsubstituted or substituted with one or twoR⁶; R² is iodo; R^(3b) is fluoro; and R^(2a), R³, R^(3a), R⁴, R⁵,R^(5a), and R⁶ are as defined in any aspect, embodiment, or claim asdescribed herein. In some embodiments, R³ and R^(3a) are each hydrogen.In some embodiments, R³ is hydrogen and R^(3a) is fluoro.

Starting from commercially-available or routinely-accessible acids offormula (109) and commercially-available or routinely-accessibleanilines of formula (105), compounds of formula (110) can be prepared bymethods apparent to those of skill in the art. Alternatively, compoundsof formula (110) can be converted to methyl ester of formula (111) bymethods known in the art. A compound of formula (110) or formula (111)is then treated with Zn(CN)₂ in the presence of a catalyst such asPd(PPh₃)₄. The compound of formula (114) or formula (115) is prepared bytreating the compound of formula (110) or formula (111) with iodine inthe presence of siver trifluoroacetate or alternatively with iodinemonochloride. In the case of the compound of formula (115), the methylester is then hydrolyzed to the corresponding acid. The acid-containingcompound of formula (114) can be activated with numerous reagentsapparent to those of skill in the art to produce compounds with asuitable leaving group attached to the carbonyl of the C(O)OH acidgroup, for example an acid chloride produced from the reaction of (114)with thionyl chloride or an active ester produced from the reaction of(114) with reagents such as EDCl or HOBt. The acid chlorides or activeesters can then be reacted with compounds of formula R⁴OH, HNR⁵R^(5a),HN(OR^(5b))R^(5a), R^(a)—H (where R^(a) is a N-linked heterocycloalkylunsubstituted or substituted with one or two R⁶), or suitable protectedforms thereof to produce compounds of formula (Ib).

Utilizing the Exemplary Preparation Schemes provided herein andprocedures known to one of ordinary skill in the art, the compounds inTables 1 and 2 can be prepared.

Example 1: 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

A solution of 4-cyano-2-fluorobenzoic acid (3.0 g, 18.1 mmol) in THF (30mL) stirred under N₂ at −78 C was treated with LDA (2.0 M in THF, 27.2mL, 54.5 mmol) added dropwise. After 20 min a solution of2-fluoro-4-iodoaniline (12.9 g, 54.5 mmol) in dry THF (15 mL) was addeddropwise and the reaction mixture was further stirred allowing it towarm up to room temperature. After 16 h the reaction mixture wasconcentrated in vacuo, acidified with 1M HCl and extracted twice withEt₂O. The combined organic phase was washed with brine, dried overNa₂SO₄, filtered, and concentrated in vacuo. The crude residue waspurified by trituration in boiling DCM to give the product (1.57 g,22.7%) as a yellow solid. m/z 381.1 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆) δppm 13.91 (s, 1H), 9.74 (s, 1H), 8.04 (d, J=8.2 Hz, 1H), 7.77 (dd,J=10.3, 2.0 Hz, 1H), 7.58 (dt, J=8.5, 1.3 Hz, 1H), 7.42-7.33 (m, 2H),7.24 (dd, J=8.2, 1.6 Hz, 1H)

Example 2: 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

A microwave vial was charged with4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (80% pure) (0.19 g,0.4 mmol), 3-hydroxyazetidine hydrochloride (0.07 g, 0.6 mmol), HATU(0.25 g, 0.6 mmol) and diisopropyl ethyl amine (140 μL, 0.8 mmol) andN,N-dimethylformamide (6 mL). The reaction mixture was stirred at roomtemperature overnight. The reaction was quenched with water andextracted with ethyl acetate. The organics were washed with water, brineand dried over sodium sulfate. The solvents were evaporated. The residuewas purified by flash chromatography (12 g silica, 0-5% methanol indichloromethane) and again by reverse phase HPLC (30-95%Acetonitrile/water) to give the product as a light yellow solid (71 mg,41%). m/z 438.1 [M+1]⁺. ¹H NMR (300 MHz, DMSO-d6): δ8.97 (s, 1H),7.69-7.65 (dd, J=1.2 Hz and 0.9 Hz, 1H), 7.55-7.47 (d, 2H), 7.40 (s,1H), 7.28-7.18 (m, 2H), 5.80 (s, 1H), 4.45 (s, 1H), 4.35 (t, J=7.6 Hz,1H), 4.21 (t, J=8.9 Hz, 1H), 4.00-3.98 (mi, 1H), 3.76-3.73 (n, 1H).

The following compounds were prepared as described in Example 2,replacing the 3-hydroxyazetidine hydrochloride with an appropriate aminewhich is commercially available or prepared using conditions known toone of ordinary skill in the art.

Ex. Comp. No. No. Structure 1H NMR (400 MHz, DMSO-d6) m/z  3 1.003

δ 7.75 (d, J = 8.1 Hz, 1H), 7.59-7.50 (m, 2H), 7.37 (s, 1H), 7.23-7.17(m, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.48 (t, J = 5.6 Hz, 2H). (CD3OD)424.2 [M −1]⁻  4 1.004

δ 7.69 (d, J = 8.1 Hz, 1H), 7.59-7.50 (m, 2H), 7.38 (s, 1H), 7.23-7.17(m, 2H), 3.64 (t, J = 6.2 Hz, 2H), 3.46 (t, J = 7.1 Hz, 2H), 1.84-1.79(m, 2H). (CD3OD) 438.2 [M −1]⁻  5 1.005

δ 9.78 (s, 1H), 8.82 (d, J = 4.2 Hz, 1H), 7.74-7.67 (m, 2H), 7.50 (d, J= 8.1 Hz, 1H), 7.41 (s, 1H), 7.30-7.25 (m, 2H), 2.85-2.81 (m, 1H),0.73-0.66 (m, 2H), 0.59-0.56 (m, 2H). 420.2 [M −1]⁻  6 1.006

δ 9.65 (s, 1H), 8.78 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H),7.51 (d, J = 8.4 Hz, 1H), 7.25 (s, 1H), 7.21-7.18 (m, 1H), 7.11 (d, J =8.1 Hz, 1H), 2.84-2.82 (m, 1H), 2.16 (s, 3H), 0.71-0.67 (m, 2H),059-0.56 (m, 2H). 416.2 [M −1]⁻  7 1.007

δ 12.05 (s, 1H), 9.26 (s, 1H), 7.69 (d, J = 10.5 Hz, 1H), 7.62 (d, J =7.5 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.40 (s, 1H), 7.31- 7.26 (m, 2H),3.69 (s, 3H). 410.2 [M −1]⁻  8 1.008

δ 11.94 (s, 1H), 9.25 (s, 1H), 7.71-7.67 (m, 2H), 7.49 (d, J = 8.1 Hz,1H), 7.40 (s, 1H), 7.32-7.23 (m, 2H), 3.94-3.86 (q, 2H), 1.18 (t, J =7.1 Hz, 3H). 424.2 [M −1]⁻  9 1.009

δ 11.93 (s, 1H), 9.23 (s, 1H), 7.69 (d, J = 10.5 Hz, 1H), 7.62 (d, J =7.5 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.39 (s, 1H), 7.32- 7.25 (m, 2H),3.81 (t, J = 6.5 Hz, 2H), 1.62- 1.55 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H).438.2 [M −1]⁻ 10 1.010

δ 7.74-7.66 (m, 2H), 7.50-7.47 (m, 1H), 7.39 (s, 1H), 7.27 (t, J = 7.4Hz, 2H), 3.90 (t, J = 4.7 Hz, 2H), 3.60 (t, J = 4.8 Hz, 2H). 440.1 [M−1]⁻ 11 1.011

δ 12.01 (s, 1H), 9.09 (s, 1H), 7.64-7.61 (m, 2H), 7.52 (d, J = 8.4 Hz,1H), 7.22-7.19 (m, 2H), 7.10 (d, J = 8.7 Hz, 1H), 3.90 (t, J = 4.7 Hz,2H), 3.61 (t, J = 5.2 Hz, 2H), 2.15 (s, 3H) 436.2 [M −1]⁻ 12 1.012

δ 12.01 (s, 1H), 9.24 (s, 1H), 7.70-7.61 (m, 2H), 7.49 (d, J = 8.1 Hz,1H), 7.38 (s, 1H), 7.31-7.22 (m, 2H), 3.68 (d, J = 7.2 Hz, 2H),1.07-1.00 (m, 1H), 0.52-0.49 (m, 2H), 0.25-0.24 (m, 2H) 450.2 [M −1]⁻ 131.013

δ 11.92 (s, 1H), 9.08 (s, 1H), 7.64-7.58 (m, 2H), 7.51 (d, J = 8.4Hz,1H), 7.21-7.19 (m, 2H), 7.09 (d, J = 9.0 Hz, 1H), 3.68 (d, J = 8.4Hz, 2H), 2.14 (s, 3H), 1.08-1.06 (m, 1H), 0.53-0.50 (m, 2H), 0.26-0.23(m, 2H) 446.3 [M −1]⁻ 14 1.014

δ 12.03 (s, 1H), 9.18 (s, 1H), 7.71-7.68 (dd, J = 1.8 Hz and 1.8 Hz,1H), 7.61 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.39 (s, 1H),7.32-7.22 (m, 2H), 4.64 (t, J = 6.8 Hz, 2H), 4.36 (t, J = 6.0 Hz, 2H),4.10 (d, J = 8.4 Hz, 2H) 466.2 [M −1]⁻ 15 1.015

δ 7.56-7.47 (m, 3H), 7.29-7.26 (m, 2H), 7.09-7.02 (m, 1H), 4.45-4.36 (m,1H), 3.69-3.45 (m, 4H), 2.07-1.95 (m, 2H). (CD3OD) 452.1 [M + 1]⁺

Example 16:N-(azetidin-3-ylmethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

Step 1: tert-butyl3-(((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)methyl)azetidine-1-carboxylate

A microwave vial was charged with4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (0.100 g, 0.3mmol), tert-butyl 3-((aminooxy)methyl)azetidine-1-carboxylate, (60%pure) (0.13 g, 0.4 mmol), HATU (0.15 g, 0.4 mmol) and diisopropyl ethylamine (135 μL, 0.8 mmol) in N,N-dimethylformamide (3 mL). The reactionmixture was stirred at room temperature overnight. The reaction wasquenched with water and extracted with ethyl acetate. The organics werewashed with water, brine and dried over sodium sulfate. The solventswere evaporated. The residue was purified by flash chromatography (12 gsilica, 0-70% ethyl acetate in hexanes). The product fractions werecollected and the solvent was removed to give a yellow oil (77 mg, 52%).m/z 565.2 [M−1]⁻.

Step 2:N-(azetidin-3-ylmethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

A round bottom flask was charged with tert-butyl3-(((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)methyl)azetidine-1-carboxylate(0.077 g, 0.1 mmol) in dichloromethane (6 mL) and hydrogen chloride (4.0M in 1,4-dioxane, 0.510 ml, 2.0 mmol) was added dropwise. The reactionmixture was stirred at room temperature overnight. The reaction mixturewas concentrated in vacuo. The residue was purified by reverse phaseHPLC ((10-95% Acetonitrile/water (0.1% TFA)). The product fractions werecollected, washed with sodium bicarbonate and dried in vacuo to give theproduct as a yellow solid. m/z 461.7 [M+1]⁺. ¹H NMR (300 MHz, CDCl3): δ8.41 (s, 1H), 7.67 (d, J=8.1 Hz, 1H), 7.50-7.42 (m, 2H), 7.35 (s, 1H),7.12-7.03 (m, 1H), 4.16-4.10 (m, 1H), 4.06-4.00 (m, 1H), 3.94-3.90 (m,1H), 3.73-3.68 (m, 1H), 2.89-2.79 (m, 3H).

Example 17: 4-Cyano-2-((2-fluoro-4-iodophenyl)amino)-N-hydroxybenzamide

A solution of hydroxylamine hydrochloride (333 mg, 0.52 mmol) in dry DMF(2.4 mL) and dry acetonitrile (2.4 mL) stirred at room temperature wastreated with Et₃N (1.33 mL, 9.6 mmol). After 1 h the suspension wasdiluted with DCM (2.4 mL), cooled down to 0° C. and a solution of4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (209 mg, 0.52mmol) in dry THF (2.4 mL) was added. After 1 h the reaction mixture wasdiluted with EtOAc, quenched with a saturated NH₄Cl aqueous solution,partitioned, and the aqueous phase was extracted with EtOAc. Thecombined organic phase was washed with brine, dried over Na₂SO₄,filtered, and concentrated in vacuo. The crude material was purified bypreparative HPLC purification to give the product (37 mg, 17.8%) as ayellow solid. m/z 396.1 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 11.60(s, 1H), 9.46 (s, 1H), 9.41 (s, 1H), 7.72 (dd, J=10.5, 2.0 Hz, 1H), 7.64(d, J=8.0 Hz, 1H), 7.56-7.49 (m, 1H), 7.46 (s, 1H), 7.37-7.28 (m, 2H).

Example 18:2-((4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethan-1-aminium2,2,2-trifluoroacetate Step 1: tert-Butyl(2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethyl)carbamate

A solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (150mg, 0.39 mmol) and HATU (298 mg, 0.78 mmol) in dry DMF (3.9 mL) wastreated with dry DIPEA (0.13 mL, 0.78 mmol). The reaction mixture wasstirred at 50° C. for 30 min, cooled down to room temperature and asolution of tert-butyl (2-(aminooxy)ethyl)carbamate (103 mg, 0.58 mmol)in dry DMF (0.5 mL) was added. After 16 h the reaction mixture wasdiluted with EtOAc, quenched with H₂O, partitioned, and the aqueousphase was extracted with EtOAc. The combined organic phase was washedwith brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Thecrude residue was purified by a flash column chromatography (Silica,1-40% EtOAc in hexanes) to give the product (87 mg, 41%) as a yellowoil. UPLC-MS (Acidic Method, 2 min): rt 1.27 min. m/z 539.1 [M−H]⁻. ¹HNMR (400 MHz, CDCl₃): δ ppm 10.56 (s, 1H), 9.45 (s, 1H), 7.59 (d, J=8.1Hz, 1H), 7.57-7.46 (m, 2H), 7.32 (s, 1H), 7.16-7.07 (m, 2H), 5.06 (s,1H), 4.01 (t, J=4.9 Hz, 2H), 3.54-3.45 (m, 2H), 1.49 (s, 9H).

Step 2:2-((4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethan-1-aminium2,2,2-trifluoroacetate

A solution of tert-butyl(2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)ethyl)-carbamate (67 mg, 0.12 mmol) in dioxane (1.0 mL) stirred at roomtemperature was treated with 4N HCl in dioxane (62 μl, 0.24 mmol). After1 h an additional portion of 4N HCl in dioxane (62 μl, 0.24 mmol) wasadded, similarly after 16 h. After 48 h a further portion of 4N HCl indioxane (0.25 mL, 1 mmol) was added. After 48 h the reaction mixture wasconcentrated in vacuo. The crude material was purified by preparativeHPLC purification to give the product (21.1 mg, 32%) as a yellow solidin a form of its trifluoroacetate salt. m/z 441.1 [M+H]⁺. ¹H NMR (400MHz, DMSO-d₆): δ ppm 7.79-7.69 (m, 2H), 7.54 (dd, J=8.3, 1.9 Hz, 1H),7.45 (d, J=1.5 Hz, 1H), 7.35 (dd, J=8.1, 1.5 Hz, 1H), 7.29 (t, J=8.6 Hz,1H), 4.07 (t, J=5.1 Hz, 2H), 3.08 (t, J=5.2 Hz, 2H).

Example 19: Methyl2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetate

A solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (500mg, 1.30 mmol) and HATU (995 mg, 2.61 mmol) in dry DMF (13.0 mL) wastreated with dry DIPEA (0.45 mL, 2.61 mmol). The reaction mixture wasstirred at 50° C. for 30 min, cooled down to room temperature and asolution of methyl 2-(aminooxy)acetate hydrochloride (270 mg, 1.96 mmol)in dry DMF (1.0 mL) was added followed by dry DIPEA (0.34 mL, 2.0 mmol).After 48 h the reaction mixture was diluted with EtOAc, quenched withH₂O, partitioned, and the aqueous phase was extracted with EtOAc. Thecombined organic phase was washed with brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The crude residue was purified by aflash column chromatography (Silica, 10-41% EtOAc in hexanes) to givethe product (373 mg, 61%) as a yellow solid. m/z 468.1 [M−H]⁻. ¹H NMR(400 MHz, DMSO-d₆): δ ppm 12.29 (s, 1H), 9.16 (s, 1H), 7.76-7.60 (m,2H), 7.52 (dt, J=8.4, 1.4 Hz, 1H), 7.41 (s, 1H), 7.35-7.19 (m, 2H),4.72-4.55 (m, 2H), 3.70 (s, 3H).

Example 20:2-((4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetic acid

A solution of methyl2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy) acetate (306mg, 0.64 mmol) in THF (1.93 mL), MeOH (0.64 mL), and H₂O (1.28 mL)stirred at room temperature was treated with 1M LiOH (0.64 mL, 0.64mmol). After 24 h an additional portion of 1M LiOH (0.64 mL, 0.64 mmol)was added and stirring continued. After 24 h the reaction mixture wasdiluted with a saturated NaHCO₃ aqueous solution, partitioned withEtOAc, and the aqueous phase was acidified with 1M HCl and extractedwith EtOAc. The organic phase was washed with 1M HCl and brine, driedover NaHCO₃, filtered and concentrated in vacuo to give the product (238mg, 81%) as a yellow solid. m/z 454.0 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆):δ ppm 13.00 (s, 1H), 12.30 (s, 1H), 9.21 (s, 1H), 7.72 (dd, J=10.4, 1.9Hz, 1H), 7.68 (s, 1H), 7.53 (dt, J=8.5, 1.4 Hz, 1H), 7.40 (d, J=1.6 Hz,1H), 7.34-7.24 (m, 2H), 4.52 (s, 2H).

Example 21:N-(2-Amino-2-oxoethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

Step 1: 2-((4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetylchloride

A solution of2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetic acid(181 mg, 0.39 mmol) in dioxane (1.5 mL) stirred at room temperatureunder N₂ was treated with SOCl₂ (0.3 mL, 3.9 mmol). After 24 h thereaction mixture was concentrated in vacuo and azeotroped with drytoluene (3×5 mL). The resultant orange solid was used in the subsequentreaction without further purification. m/z 468.1 [M+H]⁻ (detected as thecorresponding methyl ester after quenching an aliquot of the mixturewith 10% pyridine in MeOH).

Step 2:N-(2-Amino-2-oxoethoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

A solution of2-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)acetylchloride (188 mg, 0.39 mmol) in dry dioxane (0.9 mL) stirred at 0° C.under N₂ was treated with 0.5 M NH₃ in dioxane (0.91 mL). After 48 h thereaction mixture was diluted with EtOAc, quenched with 1M HCl,partitioned and the organic phase was washed with brine, dried overNa₂SO₄, filtered and concentrated in vacuo. The crude material waspurified by preparative HPLC purification to give the product (49.7 mg,28%) as a yellow solid. m/z 453.0 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δppm 12.29 (br s, 1H), 9.40 (br s, 1H), 7.74-7.69 (m, 2H), 7.54-7.48 (m,1H), 7.40 (s, 1H), 7.34-7.24 (m, 2H), 4.34 (s, 2H).

Example 22: Methyl3-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy) propanoate

A solution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (500mg, 1.30 mmol) and HATU (995 mg, 2.61 mmol) in dry DMF (13.0 mL) stirredat room temperature was treated with dry DIPEA (0.45 mL, 2.61 mmol). Thereaction mixture was stirred at 50° C. for 30 min, cooled down to roomtemperature and a suspension of methyl 3-(aminooxy)propanoatehydrochloride (305 mg, 1.96 mmol) in dry acetonitrile (2.9 mL) and dryTHF (2.9 mL) was added, followed by dry DIPEA (0.45 mL, 2.6 mmol). After16 h the reaction mixture was diluted with EtOAc, quenched with H₂O,partitioned, and the aqueous phase was extracted with EtOAc. Thecombined organic phase was washed with brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The crude residue was purified by aflash column chromatography (Silica, 20-50% EtOAc in hexanes) to givethe product (369 mg, 59%) as a yellow solid. m/z 482.0 [M−H]⁻. ¹H NMR(400 MHz, DMSO-d₆): δ ppm 12.05 (s, 1H), 9.29 (s, 1H), 7.72 (dd, J=10.5,1.9 Hz, 1H), 7.66 (s, 1H), 7.53 (d, J=8.6 Hz, 1H), 7.42 (d, J=1.7 Hz,1H), 7.36-7.25 (m, 2H), 4.14 (m, 2H), 3.61 (s, 3H), 2.70 (t, J=6.6 Hz,2H).

Example 23:3-((4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)propanoic acid

A solution of methyl3-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy) propanoate(321 mg, 0.66 mmol) in THF (4.0 mL), MeOH (1.33 mL), and H₂O (2.65 mL)stirred at room temperature was treated with 1 M LiOH (1.33 mL, 1.33mmol). After 3 days an additional portion of 1 M LiOH (0.32 mL, 0.32mmol) was added and stirring continued. After 3 h the reaction mixturewas diluted with a saturated NaHCO₃ aqueous solution, partitioned withEtOAc, and the aqueous phase was acidified and extracted with EtOAc. Thefirst EtOAc extract was washed with 1 M HCl and brine sequentially,dried over NaHCO₃, filtered and concentrated in vacuo to give theproduct (203 mg, 65.5% yield, 79% pure) as a yellow solid. The secondEtOAc extract was washed with brine, dried over NaHCO₃, filtered andconcentrated in vacuo to give the product (46 mg, 14.8% yield, 100%pure) as a yellow solid. m/z 468.1 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δppm 12.37 (m, 2H), 9.29 (s, 1H), 7.76-7.62 (m, 2H), 7.53 (dd, J=8.1, 1.9Hz, 1H), 7.42 (d, J=1.7 Hz, 1H), 7.35-7.24 (m, 2H), 4.12 (s, 2H), 2.61(t, J=6.1 Hz, 2H).

Example 24:3-((2-Fluoro-4-iodophenyl)amino)-4-(3-oxoisoxazolidine-2-carbonyl)benzonitrile

A solution of3-((4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamido)oxy)propanoic acid(203 mg, 0.43 mmol) in dioxane (1.7 mL) stirred at room temperatureunder N₂ was treated with SOCl₂ (0.32 mL, 4.3 mmol). After 24 h thereaction mixture was concentrated in vacuo and azeotroped with drytoluene (3×5 mL). The resulting orange solid was used in the next stepwithout further purification. m/z 450.0 [M+H]⁻.

Example 25:N-(3-Amino-3-oxopropoxy)-4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzamide

A solution of3-((2-fluoro-4-iodophenyl)amino)-4-(3-oxoisoxazolidine-2-carbonyl)benzonitrile(195 mg, 0.43 mmol) in dry dioxane (1.03 mL) stirred at 0° C. under N₂was treated with 0.5 M NH₃ in dioxane (0.99 mL, 0.49 mmol). After 30 minthe reaction mixture was diluted with EtOAc, quenched with 1M HCl,partitioned and the organic phase was washed with brine, dried overNa₂SO₄, filtered and concentrated in vacuo. The crude material waspurified by preparative HPLC purification to give the product (22.2 mg,11%) as a yellow solid. m/z 467.1 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δppm 12.04 (br s, 1H), 9.47 (br s, 1H), 7.71 (dd, J=10.4, 2.0 Hz, 2H),7.52 (d, J=8.0 Hz, 1H), 7.41 (s, 1H), 7.33-7.26 (m, 2H), 6.90 (br s,2H), 4.09 (t, J=5.1 Hz, 2H), 2.44 (t, J=6.3 Hz, 2H).

Example 26: 3-Hydroxycyclobutyl4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoate

Step 1: 4-Cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride(General preparation)

A suspension of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoic acid(200 mg, 0.52 mmol) in dioxane (1.61 mL) stirred at room temperature wastreated with SOCl₂ (0.38 mL, 5.23 mmol). The reaction mixture wasfurther stirred at 50° C. under N₂. After 48 h the reaction mixture wasconcentrated in vacuo and azeotroped with dry toluene (2×5 mL). Theresultant crude material in residual toluene was used in the next stepwithout further purification.

Step 2: 3-Hydroxycyclobutyl4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoate

To a solution of cyclobutane-1,3-diol (159 mg, 1.8 mmol) and Et₃N (0.16mL, 1.1 mmol) in dry THE (1.0 mL) stirred at 0° C. under N₂ atmosphere asolution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride(157 mg, 0.39 mmol) in dry THF (1.3 mL) was added. After 1 h thereaction mixture was diluted with EtOAc, quenched with a saturated NH₄Claqueous solution, partitioned, and the aqueous phase re-extracted withEtOAc. The combined organic phases were washed with brine, dried overNa₂SO₄, filtered, and concentrated in vacuo. The crude residue waspurified by a flash column chromatography (Silica, 5-31% EtOAc inhexanes) to give the product (43.7 mg, 24.7%) as a yellow solid. m/z451.1 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 9.36 (s, 1H), 8.06 (d,J=8.2 Hz, 1H), 7.76 (dd, J=10.3, 1.9 Hz, 1H), 7.58 (dt, J=8.4, 1.3 Hz,1H), 7.38-7.30 (m, 2H), 7.27 (dd, J=8.2, 1.6 Hz, 1H), 5.26 (d, J=6.5 Hz,1H), 4.74 (p, J=7.3 Hz, 1H), 3.88 (h, J=6.9 Hz, 1H), 2.86-2.70 (m, 2H),2.12-1.98 (m, 2H).

Example 27: 3-Hydroxy-2,2,4,4-tetramethylcyclobutyl4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoate

To a solution of 2,2,4,4-tetramethylcyclobutane-1,3-diol (520 mg, 3.6mmol) and Et₃N (0.32 mL, 2.3 mmol) in dry THF (2.0 mL) stirred at 0° C.under N₂ atmosphere a solution of4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride (314 mg, 0.78mmol) in dry THE (2.7 mL) was added. After 48 h the reaction mixture wasdiluted with EtOAc, quenched with a saturated NH₄Cl aqueous solution,partitioned, and the aqueous phase re-extracted with EtOAc. The combinedorganic phases were washed with brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo. The crude residue was purified by a flash columnchromatography (Silica, 0-50% EtOAc in hexanes) to give the product(26.6 mg, 6.7%) as a yellow solid. m/z 506.9 [M−H]⁻. ¹H NMR (400 MHz,DMSO-d₆): δ ppm (Note: a mixture of diastereoisomers 2:1) 9.31 (s,0.35H), 9.29 (s, 0.67H), 8.10 (d, J=8.2 Hz, 0.37H), 8.06 (d, J=8.2 Hz,0.63H), 7.77 (dd, J=10.3, 1.9 Hz, 1H), 7.59 (dd, J=7.8, 1.9 Hz, 1H),7.39-7.34 (m, 2H), 7.34-7.27 (m, 1H), 5.01 (t, J=4.8 Hz, 1H), 4.52 (s,0.35H), 4.41 (d, J=0.9 Hz, 0.67H), 3.53 (d, J=4.8 Hz, 0.36H), 3.37 (d,J=4.8 Hz, 0.66H), 1.18 (s, 4H), 1.09 (s, 2H), 1.07 (s, 2H), 1.00 (s,4H).

Example 28: 2,3-Dihydroxypropyl4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoate

To a solution of 1,2,3-propanetriol (332 mg, 3.6 mmol) and Et₃N (0.32mL, 2.3 mmol) in dry THF (2.7 mL) stirred at 0° C. under N₂ atmosphere asolution of 4-cyano-2-((2-fluoro-4-iodophenyl)amino)benzoyl chloride(314 mg, 0.78 mmol) in dry THF (2.0 mL) was added. After 16 h thereaction mixture was diluted with EtOAc, quenched with a saturated NH₄Claqueous solution, partitioned, and the aqueous phase re-extracted withEtOAc. The combined organic phases were washed with brine, dried overNa₂SO₄, filtered, and concentrated in vacuo. A portion of the crudematerial (198 mg) was purified by preparative HPLC purification to givethe product (34 mg, 9.5%) as a glassy yellow solid. m/z 455.0 [M−H]⁻. ¹HNMR (400 MHz, DMSO-d₆): δ ppm 9.33 (s, 1H), 8.12 (d, J=8.2 Hz, 1H), 7.77(dd, J=10.3, 1.9 Hz, 1H), 7.59 (dt, J=8.4, 1.4 Hz, 1H), 7.39-7.30 (m,2H), 7.29 (dd, J=8.2, 1.6 Hz, 1H), 5.12 (d, J=5.3 Hz, 1H), 4.75 (t,J=5.7 Hz, 1H), 4.37 (dd, J=11.1, 3.9 Hz, 1H), 4.23 (dd, J=11.1, 6.2 Hz,1H), 3.88-3.76 (m, 1H), 3.53-3.38 (m, 2H).

Example 29: 4-cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzoicacid

Step 1: 2-fluoro-4-(trimethylsilyl)aniline

A round bottom flask was charged with 4-bromo-2-fluoroaniline (1.00 g,5.3 mmol) and anhydrous tetrahydrofuran (8 mL), cooled to −78° C. and a2.5M solution of nBuLi in hexanes (8 ml, 20 mmol) was added dropwisekeeping the internal temperature below −60° C. The reaction mixture wastreated dropwise with chlorotrimethylsilane (2.26 ml, 17.4 mmol.),keeping the internal temperature below −60° C. The reaction mixture wasallowed to warm to 0° C. over one hour period. The reaction mixture waspoured into ice-cold 2M hydrochloric acid and was vigorously stirred for10 minutes. The organic layer was separated and the aqueous layer wasextracted with ethyl acetate. The combined organics were dried overmagnesium sulfate and evaporated to dryness to give the product ascolorless oil (0.58 g, 54%). m/z 184.2 [M+1]*.

Step 2:4-bromo-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoicacid

To a stirred solution comprised of 2-fluoro-4-(trimethylsilyl)aniline(1.0 g, 5.1 mmol) in tetrahydrofuran (6 mL) at −78° C. was added lithiumdiisopropylamide (2.0 M in THF/heptane/ethylbenzene, 2.5 ml, 5.1 mmol).The resulting suspension was stirred vigorously for 10 minutes, afterwhich time a solution of 4-bromo-2,3-difluorobenzoic acid (0.400 g, 1.7mmol) in tetrahydrofuran (5 mL) was added. The cold bath wassubsequently removed, and the reaction mixture was stirred at roomtemperature overnight. The mixture was concentrated and the concentratewas treated with 3M hydrochloric acid (10 mL). The resulting suspensionwas extracted with ethyl ether. The combined organics were dried oversodium sulfate. The solvents were removed in vacuo. Hexanes was addedinto the residue. The precipitated beige solid (382 mg) was washed usinghexane and a few drop of ethyl acetate and isolated by filtration. m/z400.1 [M+]. ¹H NMR (300 MHz, DMSO-d₆): δ 9.06 (s, 1H), 7.70-7.66 (m,1H), 7.38-7.33 (m, 1H), 7.27 (d, J=11.7 Hz, 1H), 7.20-7.17 (m, 1H),6.93-6.86 (m, 1H), 0.22 (s, 9H).

Step 3:4-cyano-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoicacid

A microwave vial was charged with4-bromo-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoicacid (0.180 g, 0.4 mmol), zinc cyanide (0.05 g, 0.4 mmol) andtetrakis(triphenylphosphine)palladium(0) (0.05 g, 0.05 mmol) inN,N-dimethyl formamide (3 mL) under argon. The reaction mixture wasstirred at 90° C. overnight. The reaction was quenched with water andextracted with ethyl acetate. The organics were washed with water, brineand dried over sodium sulfate. The solvents were evaporated. The residuewas purified by flash chromatography (12 g silica, 0-70% ethyl acetatein hexanes) to give the product as a yellow solid (50 mg, 32%). ¹H NMR(300 MHz, CDCl3): δ 8.95 (s, 1H), 7.93 (d, J=8.4 Hz, 1H), 7.24-7.21 (m,2H), 7.19-7.08 (m, 1H), 7.00-6.93 (m, 1H), 0.28 (s, 9H).

Step 4: 4-cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid

A round bottom flask was charged with4-cyano-3-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)benzoicacid (0.49 g, 1.4 mmol) in anhydrous methanol (6 mL) and anhydrousdichloromethane (6 mL). Silver trifluoroacetate (0.66 g, 3.0 mmol) wasadded. The reaction mixture was cooled to 0° C. Iodine (0.72 g, 2.8mmol) was then added in one portion. The reaction mixture was stirred at0° C. for 2 hours. The reaction mixture was filtered through celite andthe solvents were removed. The residue was treated with saturated sodiumthiosulfate and extracted with ethyl acetate. The combined organics weredried over sodium sulfate. The solvents were removed in vacuo.Dichloromethane was added to the residue whereby a solid precipitatedout. The solid was collected by filtration and dried to give the productas beige colored solid (200 mg, 35%). ¹H NMR (300 MHz, DMSO-d₆): δ7.83-7.81 (m, 1H), 7.59-7.54 (dd, J=1.8 and 1.8 Hz, 1H), 7.39-7.36 (m,2H), 6.74-6.66 (m, 1H).

Example 30:4-cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)benzamide

A microwave vial was charged with4-cyano-3-fluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (0.100 g,0.2 mmol), 2-(aminooxy)ethanol (0.03 g, 0.4 mmol), HATU (0.14 g, 0.4mmol) and diisopropyl ethyl amine (86 μL, 0.5 mmol.) andN,N-dimethylformamide (3 mL). The reaction mixture was stirred at roomtemperature overnight. The reaction was quenched with water andextracted with ethyl acetate. The organics were washed with water, brineand dried over sodium sulfate. The solvents were evaporated. The residuewas purified by flash chromatography (12 g silica, 0-5% methanol indichloromethane). The product fractions were collected and the solventwas removed. The residue was purified again by reverse phase HPLC(20-80% Acetonitrile/water) to give the product as a light yellow solid.m/z 458.0 [M−1]⁻. ¹H NMR (300 MHz, DMSO-d₆): δ 11.93 (s, 1H), 8.44 (s,1H), 7.67-7.62 (m, 1H), 7.56 (d, J=9.6 Hz, 1H), 7.47-7.44 (m, 1H), 7.35(d, J=8.4 Hz, 1H), 6.70-6.63 (m, 1H), 3.79 (t, J=4.4 Hz, 2H), 3.55-3.53(m, 2H).

The following compounds are prepared as described in Example 30,replacing the 2-(aminooxy)ethanol with an appropriate amine which iscommercially available or prepared using conditions known to one ofordinary skill in the art.

Ex. Comp. Structure No. No. 1H NMR (300 MHz, DMSO-d6) m/z 31 1.031

δ 7.65-7.54 (m, 2H), 7.46-7.43 (m, 1H), 7.35 (d, J = 9.0 Hz, 1H),6.70-6.66 (m, 1H), 3.55 (d, J = 6.9 Hz, 2H), 1.02-1.01 (m, 1H),0.50-0.46 (m, 2H), 0.21-0.19 (m, 2H). 468.2 [M − 1]⁻

Example 32: Methyl 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinate

Step 1: 6-Chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinicacid

To a solution of 2-fluoro-4-(trimethylsilyl)aniline (9.2 g, 50 mmol) indry THF (40 mL) stirred at −78° C. was added LiHMDS (1M in THF, 100 mL,100 mmol). The reaction mixture was stirred for 1 h at −78° C. followedby an addition of a solution of 2,6-dichloroisonicotinic acid (8 g, 41.7mmol) in dry THF (31 mL). The reaction mixture was stirred at −78° C.for 1 h and then gradually warmed up to room temperature. The reactionmixture was quenched with a saturated NH₄Cl aqueous solution (100 mL) at0° C., diluted with EtOAc (200 mL), acidified with 1M HCl to pH 3 andpartitioned. The aqueous phase was extracted with EtOAc (2×100 mL), theorganic phase was washed with brine (100 mL), dried over Na₂SO₄ andconcentrated in vacuo. The crude material was purified by triturationwith methanol to give the product (10.6 g, 75%) as a yellow solid. m/z339.1/341.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 13.94 (s, 1H),10.79 (d, J=2.9 Hz, 1H), 8.41 (t, J=8.1 Hz, 1H), 8.27 (d, J=8.1 Hz, 1H),7.37 (m, 2H), 7.00 (d, J=8.1 Hz, 1H), 0.26 (s, 9H).

Step 2: Methyl6-chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

A solution of6-chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinic acid (0.5g, 1.476 mmol) in DCM (7.4 mL) stirred at room temperature was treatedwith DIPEA (0.26 mmol, 1.476 mmol). After 10 min the reaction mixturewas cooled down to 0° C. and was treated with DMF (0.03 mL) and oxalylchloride (0.12 mL, 1.476 mmol) with subsequent warm up to roomtemperature. The reaction mixture was stirred for 30 minutes, thenslowly added to the solution of DIPEA (0.26 mL, 1.476 mmol) in MeOH (7.4mL) stirred at 0° C. with a subsequent warm up to room temperature.After 15 minutes the reaction mixture was concentrated in vacuo. Theresidue was dissolved in EtOAc (20 mL), washed with a saturated NaHCO₃aqueous solution (10 mL), H₂O (10 mL), brine (10 mL), dried over Na₂SO₄and concentrated in vacuo to give the product (0.45 g, 87%) as a brownsolid. m/z 353.1/355.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 10.48(d, J=3.0 Hz, 1H), 8.36 (t, J=7.9 Hz, 1H), 8.29 (d, J=8.2 Hz, 1H),7.43-7.33 (m, 2H), 7.02 (d, J=8.2 Hz, 1H), 3.91 (s, 3H), 0.26 (s, 9H).

Step 3: Methyl6-cyano-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

A degassed solution of methyl6-chloro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino) nicotinate (0.4 g,1.13 mmol), zinc cyanide (0.11 g, 0.96 mmol) andtetrakis(triphenylphosphine) palladium(0) (0.13 g, 0.11 mmol) in NMP(3.5 mL) was heated in a microwave oven at 190° C. for 15 min. Thereaction mixture was filtered, diluted with EtOAc (20 mL), washed with asaturated NaHCO₃ aqueous solution (10 mL), H₂O (10 mL), brine (10 mL),dried over Na₂SO₄ and concentrated in vacuo. The crude material waspurified by flash column chromatography (Silica, 0-15% EtOAc in hexanes)to give the product (0.133 g, 43%) as a yellow solid. m/z 344.1 [M+H]⁺.¹H NMR (400 MHz, DMSO-d₆): δ 10.43 (d, J=2.8 Hz, 1H), 8.46 (d, J=7.8 Hz,1H), 8.32 (t, J=8.0 Hz, 1H), 7.54 (d, J=7.8 Hz, 1H), 7.45-7.36 (m, 2H),3.94 (s, 3H), 0.26 (s, 9H).

Step 4: Methyl 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinate

A suspension of silver tetrafluoroborate (85 mg, 0.437 mmol) in DCM (0.5mL) was stirred at −50° C. for 5 min in the dark, then a solution ofmethyl 6-cyano-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)-nicotinate(50 mg, 0.146 mmol) in DCM (1 mL) was added dropwise. After 15 min thereaction mixture was treated with iodine monochloride (26 mg, 0.161mmol) in DCM (0.3 mL). The reaction mixture was stirred for 15 min andan additional portion of iodine monochloride (7 mg, 0.044 mmol) in DCM(0.1 mL) was added. After 15 min the reaction mixture was quenched witha saturated Na₂S₂O₃ aqueous solution (1 mL), extracted with EtOAc (3×5mL), the organic phase was washed with brine (5 mL), dried over Na₂SO₄and the solvent was removed in vacuo to give the product (53 mg, 91%) asa yellow solid. m/z 398.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 10.37(d, J=2.8 Hz, 1H), 8.46 (d, J=7.8 Hz, 1H), 8.11 (t, J=8.6 Hz, 1H), 7.76(dd, J=10.6, 2.0 Hz, 1H), 7.64 (dd, J=8.6, 1.7 Hz, 1H), 7.55 (d, J=7.8Hz, 1H), 3.94 (s, 3H).

Example 33: 6-Cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid

To a suspension of methyl6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinate (0.42 g, 1.06 mmol)in THF (3.2 mL), MeOH (1.05 mL) and H₂O (2.1 mL) stirred at roomtemperature was treated with 1M LiOH aqueous solution (1.06 mL, 1.06mmol). After 30 min an additional portion of 1M LiOH aqueous solution(1.06 mL, 1.06 mmol) was added. After 30 min the reaction mixture wasconcentrated in vacuo, the residue was partitioned between EtOAc (10 mL)and H₂O (10 mL). The aqueous phase was acidified to pH 3 with 1M HClaqueous solution and extracted with EtOAc (3×10 mL). The combinedorganic phase was washed with brine (10 mL), dried over Na₂SO₄ andconcentrated in vacuo to give the product (0.40 g, quant.) as a yellowsolid. m/z 382.0 [M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 8.42 (d, J=7.7Hz, 1H), 8.21 (t, J=8.5 Hz, 1H), 7.73 (dd, J=10.8, 1.9 Hz, 1H), 7.62 (d,J=8.6 Hz, 1H), 7.52 (d, J=7.7 Hz, 1H).

Example 34:6-Cyano-N-ethoxy-2-((2-fluoro-4-iodophenyl)amino)nicotinamide

To a suspension of 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinicacid (100 mg, 0.26 mmol) in dry DMF (2.5 mL) were added HATU (200 mg,0.52 mmol) and DIPEA (0.13 mL, 0.65 mmol) and the reaction mixture washeated at 50° C. for 30 min. The reaction mixture was then cooled downto room temperature and a solution of ethoxyamine hydrochloride (38 mg,0.39 mmol) in dry DMF (0.5 mL) was pre-treated with DIPEA (0.05 mL, 0.39mmol) for 5 min was added. After 1 h the reaction mixture wasconcentrated in vacuo, the residue was dissolved in EtOAc (5 mL), washedwith H₂O (5 mL) and brine (5 mL). The organic phase was dried overNa₂SO₄ and concentrated in vacuo. The crude material was purified byflash column chromatography (Silica, 0-37% EtOAc in hexanes) followed bya trituration with diethyl ether to give the product (7 mg, 6%) as ayellow solid. UPLC-MS (Acidic Method, 2 min): rt 1.26 min, m/z 425.1[M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 12.18 (s, 1H), 10.89 (s, 1H),8.16 (d, J=7.7 Hz, 2H), 7.71 (dd, J=10.7, 2.0 Hz, 1H), 7.60 (dt, J=8.6,1.5 Hz, 1H), 7.55 (d, J=7.8 Hz, 1H), 4.00 (d, J=7.1 Hz, 2H), 1.24 (t,J=7.0 Hz, 3H).

Example 35: 6-Cyano-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)nicotinamide

To a solution of 6-cyano-2-((2-fluoro-4-iodophenyl)amino)nicotinic acid(100 mg, 0.261 mmol) and HATU (198.5 mg, 0.522 mmol) in DMF (3 mL)stirred at room temperature DIPEA (91 μl, 0.522 mmol) was added dropwiseand the reaction was monitored towards completion of HATU-activation ofthe acid. After 2 h 2-(aminooxy)ethan-1-ol (30.2 mg, 0.395 mmol) wasadded to the reaction mixture and it was stirred at room temperature for45 min. The reaction mixture was quenched with H₂O (30 mL) and extractedwith EtOAc (4×20 mL). The combined organic phase was washed with brine(2×20 mL), dried over Na₂SO₄, and concentrated in vacuo. The crude waspurified by preparative HPLC to give the product (55.2 mg, 48%) as ayellow solid. m/z 443.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 12.29(br s, 1H), 10.73 (s, 1H), 8.16 (d, J=7.8 Hz, 1H), 8.14 (t, J=8.5 Hz,1H), 7.71 (dd, J=10.7, 2.0 Hz, 1H), 7.60 (dt, J=8.6, 1.3 Hz, 1H), 7.55(d, J=7.8 Hz, 1H), 4.79 (br s, 1H), 4.00 (t, J=4.8 Hz, 2H), 3.66 (t,J=4.9 Hz, 2H).

Example 36: Methyl6-cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinate

Step 1:6-Chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinicacid

A solution of 2-fluoro-4-(trimethylsilyl)aniline (8.4 g, 45.7 mmol) indry THF (34 mL) stirred at −78° C. was treated with LiHMDS (1M in THF,91.2 mL, 91.2 mmol). The reaction mixture was stirred for 1 h at −78° C.and then a solution of 2,6-dichloro-5-fluoroisonicotinic acid (8 g, 38mmol) in dry THF (30 mL). The reaction mixture was stirred at −78° C.for 1 h, then gradually warmed up to room temperature and stirred for 1h. The reaction mixture was cooled down to 0° C. and was quenched with asaturated NH₄Cl aqueous solution, then diluted with EtOAc (200 mL),acidified with 1M HCl to pH 3, partitioned and the aqueous phase wasextracted with EtOAc (2×100 mL). The organic phase was washed with brine(100 mL), dried over Na₂SO₄ and concentrated in vacuo. The crudematerial was purified by trituration with methanol to the product (7.5g, 55%) as a yellow solid. m/z 357.0/359.0 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.69 (s, 1H), 8.36 (t, J=8.0 Hz, 1H), 8.28 (d, J=8.5 Hz,1H), 7.41-7.32 (m, 2H), 0.25 (s, 9H).

Step 2: Methyl6-chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

A suspension of6-chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino) nicotinicacid (2.4 g, 6.726 mmol) and DMF (0.32 mL) in DCM (32 mL) stirred at 0°C. was treated with oxalyl chloride (2.85 mL, 33.63 mmol), then stirredat reflux for 1 h. The reaction mixture was concentrated in vacuo,azeotroped with toluene (3×25 mL) and then the residue was treated withice-cold methanol (32 mL). The resulted suspension was stirred atreflux. After 2 h the reaction mixture was cooled down and formedprecipitate was filtered under reduced pressure. The collectedprecipitate was washed with ice-cold methanol (3×5 mL) and dried invacuo to give the product (2.18 g, 88%) as a yellow solid. m/z371.1/373.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.33 (d, J=3.0 Hz,1H), 8.37-8.25 (m, 2H), 7.44-7.33 (m, 2H), 3.92 (s, 3H), 0.26 (s, 9H).

Step 3: Methyl6-cyano-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate

A degassed solution of methyl6-chloro-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)amino)nicotinate(0.5 g, 1.35 mmol), zinc cyanide (0.14 g, 1.215 mmol) andtetrakis(triphenylphosphine)palladium(0) (0.23 g, 0.20 mmol) in NMP (4mL) was heated in a microwave oven at 150° C. for 15 min. The reactionmixture was filtered, diluted with EtOAc (20 mL), washed with asaturated NaHCO₃ aqueous solution (20 mL) and partitioned. The aqueousphase was extracted with EtOAc (3×10 mL) and the combined organic phasewas washed with H₂O (20 mL), brine (20 mL), dried over Na₂SO₄ andconcentrated in vacuo. The crude material was purified by flash columnchromatography (Silica, 0-15% EtOAc in hexanes) to give the product(0.425 g, 87%) as a yellow solid. m/z 362.1 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.29 (d, J=2.8 Hz, 1H), 8.51 (d, J=8.7 Hz, 1H), 8.28 (t,J=8.0 Hz, 1H), 7.46-7.37 (m, 2H), 3.96 (s, 3H), 0.27 (s, 9H).

Step 4: Methyl6-cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinate

A suspension of silver tetrafluoroborate (0.69 g, 3.531 mmol) in DCM(4.2 mL) was stirred at −50° C. for 10 min in the dark, then a solutionof methyl6-cyano-5-fluoro-2-((2-fluoro-4-(trimethylsilyl)phenyl)-amino)nicotinate(0.425 g, 1.177 mmol) in DCM (8.5 mL) was added dropwise. After 30 minthe reaction mixture was treated with iodine monochloride (0.21 g, 1.295mmol) in DCM (2.5 mL). The reaction mixture was stirred for 30 min andan additional portion of iodine monochloride (0.21 g, 1.295 mmol) in DCM(2.5 mL) was added. After 30 min the reaction mixture was quenched witha saturated Na₂S₂O₃ aqueous solution (10 mL), extracted with EtOAc (3×25mL), the organic phase was washed with brine (25 mL), dried over Na₂SO₄and the solvent was removed in vacuo to give the product (0.36 g, 76%)as a yellow solid. m/z 416.4 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ ppm10.22 (s, 1H), 8.50 (d, J=8.8 Hz, 1H), 8.05 (t, J=8.6 Hz, 1H), 7.76 (dd,J=10.6, 2.0 Hz, 1H), 7.66-7.60 (m, 1H), 3.95 (s, 3H).

Example 37: 6-Cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinicacid

To a suspension of methyl6-cyano-5-fluoro-2-((2-fluoro-4-iodophenyl)amino)nicotinate (0.36 g,0.898 mmol) in THE (2.7 mL), MeOH (0.9 mL) and H₂O (1.8 mL) stirred atroom temperature was treated with 1M LiOH aqueous solution (0.9 mL,0.898 mmol). After 30 min the reaction mixture was concentrated invacuo, the residue was partitioned between EtOAc (10 mL) and H₂O (10mL). The aqueous phase was acidified to pH 3 with 1M HCl aqueoussolution and extracted with EtOAc (3×10 mL). The combined organic phasewas washed with brine (10 mL), dried over Na₂SO₄ and concentrated invacuo to give the product (0.30 g, 86%) as a brown solid. m/z 400.0[M−H]⁻. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 10.70 (s, 1H), 8.43 (d, J=8.7Hz, 1H), 8.13 (t, J=8.7 Hz, 1H), 7.72 (dd, J=10.7, 1.9 Hz, 1H), 7.61(dt, =8.6, 1.6 Hz, 1H).

Example 38: MEK Inhibition Assay-1

The following procedure can be used to measure biochemical activity.MEK1 inhibitory activity of compounds were tested using the followingprocedure. See Anastassiadis T, et al. Comprehensive assay of kinasecatalytic activity reveals features of kinase inhibitor selectivity. NatBiotechnol. 2011, 29(11), 1039-45.

Reagents:

-   -   Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM EGTA,        0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO    -   Enzyme: MEK1, Invitrogen cat #PV3303    -   N-terminal His-tagged recombinant human full length protein,        expressed in insect cells. Activated in vitro by RAF1. MW=49.2        kDa, GenBank Accession No. NP_002746.    -   Substrate: 5 μM ERK2 (K52R),    -   Kinase-dead mutant, (GenBank Accession No. NM_0011949), aa2-358        with N-terminal His6 tag, MW=43.63 kDa, expressed in E. coli.

The substrate was prepared in freshly prepared Reaction Buffer. Thekinase was delivered into the substrate solution and gently mixed. Testcompounds were delivered in 100% DMSO into the kinase reaction mixtureby Acoustic technology (Echo550; nanolitter range), and incubated for 20min at room temperature. ³³P-ATP was delivered into the reaction mixtureto initiate the reaction. The reaction mixture was incubated for 2 hoursat room temperature. Kinase activity was detected by P81 filter-bindingmethod.

Example 39: MEK Inhibition Assay-2

MEK1 inhibitory activity of compounds were tested using the followingprocedure (protocol available atthermofisher.com/content/dam/LifeTech/migration/files/drug-discovery/pdfs.par.60256.file.dat/20130430%20ssbk%20customer %20protocol %20and %20assa y %20conditions.pdf). The Z′-LYTEbiochemical assay (ThermoFisher) employs a fluorescence-based,coupled-enzyme format and is based on the differential sensitivity ofphosphorylated and non-phosphorylated peptides to proteolytic cleavage.

Test compounds in 100% DMSO were screened in 1% DMSO (final) in thewell. For 10 point titrations, 3-fold serial dilutions are conductedfrom the starting concentration of 30 μM.

The peptide/kinase, MAP2K1 (MEK1)/inactive MAPK1 (ERK2)/Ser/Thr 03,mixture (“Peptide/kinase Mixture”) was diluted to a 2× workingconcentration in the following buffer (“Kinase Buffer”): 50 mM HEPES pH7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL kinasereaction consisted of 0.06-0.25 ng MAP2K1 (MEK1), 105 ng inactive MAPK1(ERK2), and 2 μM Ser/Thr 03 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mMMgCl₂, 1 mM EGTA. After the 1 hour incubation, 5 μL of a 1:1024 dilutionof Development Reagent A (available from Invitrogen, catalog no. PV3295)was added.

ATP solutions were diluted to a 4× working concentration in KinaseBuffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA). ATPKm apparent was previously determined using a Z′-LYTE assay. TheDevelopment Reagent was diluted in Development Buffer (available fromInvitrogen, catalog no. P3127).

Assay Protocol: 2.5 μL of 4× test compound or 100 nL of 100× TestCompound plus 2.4 μL Kinase Buffer, 5 μL of the 2× Peptide/KinaseMixture, 2.5 μL of 4×ATP Solution were added to the plates and placed ona shake plate for 30-seconds. The kinase reaction was allowed to proceedfor 60-minute at room temperature, before 5 μL of Development ReagentSolution was added, and the mixture agitated for 30-seconds on a shakeplate. The mixture was incubated for 60-minute at room temperature.Fluorescence was measured using a plate reader and the data wereanalyzed.

The maximum emission ratio was established by the 0% PhosphorylationControl (100% Inhibition Control), which contained no ATP and thereforeexhibited no kinase activity. This control yielded 100% cleaved peptidein the Development Reaction. The 100% Phosphorylation Control, whichconsisted of a synthetically phosphorylated peptide of the same sequenceas the peptide substrate, was designed to allow for the calculation ofpercent phosphorylation. This control yielded a very low percentage ofcleaved peptide in the Development Reaction. The 0% Phosphorylation and100% Phosphorylation Controls allow for the calculation of the percentphosphorylation achieved in a specific reaction well. Control wells didnot include any kinase inhibitors.

The minimum emission ratio in a screen was established by the 0%Inhibition Control, which contained active kinase. This control wasdesigned to produce a 10-50% phosphorylated peptide in the KinaseReaction. Cascade assays may produce up to 70% phosphorylated peptide.

A known inhibitor control standard curve, 10 point titration, was runfor each individual kinase on the same plate as the kinase to ensure thekinase was inhibited within an expected IC₅₀ range previouslydetermined.

The following controls are prepared for each concentration of TestCompound assayed. The Development Reaction Interference was establishedby comparing the Test Compound Control wells that did not contain ATPversus the 0% Phosphorylation Control (which did not contain the TestCompound). The expected value for a non-interfering compound should be100%. Any value outside of 90% to 110% was flagged. The Test CompoundFluorescence Interference was determined by comparing the Test CompoundControl wells that did not contain the Kinase/Peptide Mixture (zeropeptide control) versus the 0% Inhibition Control. The expected valuefor a non-fluorescence compound should be 0%. Any value>20% was flagged.

The data in Table A was calculated. XLfit from IDBS was used. The doseresponse curve was curve fit to model number 205 (sigmoidaldose-response model). If the bottom of the curve did not fit between−20% & 20% inhibition, it was set to 0% inhibition. If the top of thecurve did not fit between 70% and 130% inhibition, it was set to 100%inhibition.

TABLE A Equation Correction for Background Fluorescence FI_(Sample) -FI_(TCFI Ctl) Emission Ratio (using values corrected for backgroundfluorescence)$\frac{{{Coumarin}\mspace{14mu}{Emission}\mspace{14mu}\left( {445\mspace{14mu}{nm}} \right)}\mspace{14mu}}{{Fluorescein}\mspace{14mu}{Emission}\mspace{14mu}\left( {520\mspace{14mu}{nm}} \right)}$% Phosphorylation (% Phos)$\left\{ {1 - \frac{\left( {{Emission}\mspace{14mu}{Ratio} \times F_{100\%}} \right) - C_{100\%}}{\left( {C_{0\%} - C_{100\%}} \right) + \left\lbrack {{Emission}\mspace{14mu}{Ratio} \times \left( {F_{100\%} - F_{0\%}} \right)} \right\rbrack}} \right\}*100$% Inhibition$\left\{ {1 - \frac{\%\mspace{14mu}{Phos}_{Sample}}{\%\mspace{14mu}{Phos}_{0\%\mspace{14mu}{Inhibition}\mspace{14mu}{Ctl}}}} \right\}*100$Z′ (using Emission Ratio values)$1 - \frac{{3*{Stdev}_{0\%\mspace{14mu}{Phos}\mspace{14mu}{Ctl}}} + {3*{Stdev}_{0\%\mspace{14mu}{Inhibition}}}}{{Mean}_{0\%\mspace{14mu}{Phos}\mspace{14mu}{Ctl}} - {Mean}_{0\%\mspace{14mu}{Inhibition}}}$Difference Between Data Points |% Inhibition_(Point 1) - %Inhibition_(Point 2)| (single point only) Development ReactionInterference (DRI) (no ATP control)$\frac{{Emission}\mspace{14mu}{Ratio}_{{DRI}\mspace{14mu}{Ctl}}}{{Emission}\mspace{14mu}{Ratio}_{0\%\mspace{14mu}{Phos}\mspace{14mu}{Ctl}}}$Test Compound Fluorescence Interference (TCFI)$\frac{{FI}_{{TCFI}\mspace{14mu}{Ctl}}}{{FI}_{0\%\mspace{14mu}{Inhibitor}\mspace{14mu}{Ctl}}}$(check both Coumarin and Fluorescein emissions) FI = FluorescenceIntensity C_(100%) = Average Coumarin emission signal of the 100% Phos.Control C_(0%) = Average Coumarin emission signal of the 0% Phos.Control F_(100%) = Average Fluorescein emission signal of the 100% Phos.Control F_(0%) = Average Fluorescein emission signal of the 0% Phos.Control DRI = Development Reaction Interference TCFI = Test CompoundFluorescence Interference

Table 3 lists the MEK1 inhibition assay results of selected compoundsaccording to the above procedure. A indicates an IC₅₀ of less than orequal to 150 nM, B indicates an IC₅₀ of greater than 150 nM and lessthan or equal to 1.5 μM, and C indicates an IC₅₀ of greater than 1.5 μM.

TABLE 3 MEK1 Inhibition Assay Results Compound No. IC₅₀ against MEK11.001 B 1.005 C 1.007 B 1.008 B 1.009 B 1.010 A 1.011 B 1.012 B 1.013 B1.017 A 1.018 B 1.022 C 1.030 A 1.031 A

Example 40: Cell-Based Assay-1

Preparation of cell lines useful for testing the soft MEK inhibitors inNF1 related cell-proliferation assays can be found in Basu et al. Nature356: 713-715, 1992; and DeClue et al. Cell 69: 265-273, 1992. Inaddition, exemplary in vitro and in vivo models to determine efficacy ofthe soft MEK inhibitors described herein can be found in U.S. Pat. Nos.8,211,875 and 8,487,004, which are incorporated by reference in theirentireties.

Example 41: Cell-Based Assay-2

Alternatively, the following procedure can be used to measure cell-basedactivity. Test compounds were dissolved in DMSO in 10 mM stock. CellTiter-Glo® 2.0 Luminescent cell viability assay reagent was purchasedfrom Promega (Madison, Wis.). A375 and HCT116 cell lines were purchasedfrom American Type Culture Collection (Manassas, Va.). For A375 cells,cell culture media was DMEM+10% FBS. Cell culture media are listed inthe following table. For HCT116 cells, cell culture media was McCoy's5A+10% FBS. All media were supplemented with 100 μg/mL of penicillin,and 100 μg/mL of streptomycin. Cultures were maintained at 37° C. in ahumidified atmosphere of 5% CO₂ and 95% air.

Test compounds were diluted in DMSO solution with 10-dose and 3-folddilutions in a source plate starting at 10 mM. 25 nL of each testcompound was delivered from the source plate to each well of the384-well cell culture plates (T=Final) by Echo 550. 25 μL of culturemedium containing 2000 of A375 or HCT116 cells was added to each of thewells in duplicates of the cell culture plates (T=0 and T=Final). 25 μLof Cell Titer Glo 2.0 reagent was added to each well of cell cultureplate (T=0). The contents were mixed on an orbital shaker for 2 min andincubated at room temperature for 15 min to stabilize luminescentsignal. Luminescence was recorded by Envision 2104 Multilabel Reader(PerkinElmer, Santa Clara, Calif.). The number of viable cells inculture was determined based on quantitation of the ATP present in eachculture well. The cells in cell culture plate (T=Final) were incubatedwith the compounds at 37° C., 5% CO₂ for 72 hours. 25 μL of Cell TiterGlo 2.0 reagent was added to each well. The contents were mixed on anorbital shaker for 2 min and incubated at room temperature for 15 min tostabilize luminescent signal. Luminescence was recorded by Envision 2104Multilabel Reader (PerkinElmer, Santa Clara, Calif.). The number ofviable cells in culture was determined based on quantitation of the ATPpresent in each culture well. The GI₅₀ curves were plotted using theGraphPad Prism 4 program based on a sigmoidal dose-response equationY=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogEC50−X)*HillSlope)). All parameters in the equation were calculated byGraphPad Prism 4 program. GI₅₀ is the concentration of the compoundcalculated according to [(T_(i)−T_(z))/(C−T_(z))]*100=50 where T_(i) isthe row data of cells with test compounds at T=Final; T_(z) is the rowdata of cells without compounds at T=0 h; C is the row data of cellswith control compound staurosporine (Sigma-Aldrich) at T=72 h.Accordingly, GI₅₀ is the value of 10^(X), where X was calculated by theCurve Fitting Equation when Y=50 using Excel.

Example 42: S9 Stability Assays

Compounds can be assessed for metabolic stability in human skin byassessing their rate of disappearance from human S9 skin fraction.Similarly, compounds can be assessed for metabolic stability in humanliver by assessing their rate of disappearance from human S9 liverfraction. The protocol below is used to assess the difference betweenskin and hepatic metabolism.

The assay was carried out in 96-well microtiter plates at 37° C.Reaction mixtures (25 μL) contained a final concentration of 1 μM testcompound, 2 mg/mL liver or skin protein, and 1 mM NADPH in buffer (100mM potassium phosphate, pH 7.4 buffer with 1 mM EDTA, 3 mM MgCl₂). Ateach of the time points (0, 15, 30, and 60 minutes), 150 μL of quenchsolution (100% acetonitrile with 0.1% formic acid) with internalstandard was transferred to each well. Besides the zero minute controls,mixtures containing the same components except the NADPH were alsoprepared as the negative control. Verapamil or testosterone was includedas a positive control to verify assay performance. Plates were sealedand centrifuged at 4° C. for 15 minutes at 4000 rpm. The supernatant wastransferred to fresh plates for LC/MS/MS analysis.

All samples were analyzed on LC/MS/MS using an AB Sciex API 4000instrument, coupled to a Shimadzu LC-20AD LC Pump system. Analyticalsamples were separated using a Waters Atlantis T3 dC18 reverse phaseHPLC column (20 mm×2.1 mm) at a flow rate of 0.5 mL/min. The mobilephase consisted of 0.1% formic acid in water (solvent A) and 0.1% formicacid in 100% acetonitrile (solvent B).

The extent of metabolism was calculated as the disappearance of the testcompound, compared to the 0-min control reaction incubations. Initialrates were calculated for the compound concentration and used todetermine t_(1/2) values and subsequently, the intrinsic clearance,CL_(int)=(0.693) (1/t_(1/2) (min)) (mL incubation/mg of S9 protein).

Example 43: Microsomal Stability Assay

Metabolic stability of testing compound can be evaluated using humanliver microsomes to predict intrinsic clearance. Human liver microsomesare obtained from Corning Gentest.

The assay was carried out in 96-well microtiter plates at 37° C.Reaction mixtures (25 μL) contain a final concentration of 1 μM testcompound, 0.5 mg/mL liver microsomes protein, and 1 mM NADPH in buffer(100 mM potassium phosphate, pH 7.4 buffer with 3 mM MgCl₂). At each ofthe time points (0, 15, 30, and 60 minutes), 150 μL of quench solution(100% acetonitrile with 0.1% formic acid) with internal standard wastransferred to each well. Verapamil was included as a positive controlto verify assay performance. Plates were sealed and centrifuged at 4° C.for 15 minutes at 4000 rpm. The supernatant was transferred to freshplates for LC/MS/MS analysis.

All samples were analyzed on LC/MS/MS using an AB Sciex API 4000instrument, coupled to a Shimadzu LC-20AD LC Pump system. Analyticalsamples were separated using a Waters Atlantis T3 dC18 reverse phaseHPLC column (20 mm×2.1 mm) at a flow rate of 0.5 mL/min. The mobilephase consisted of 0.1% formic acid in water (solvent A) and 0.1% formicacid in 100% acetonitrile (solvent B).

The extent of metabolism was calculated as the disappearance of the testcompound, compared to the 0-min time incubation. Initial rates werecalculated for the compound concentration and used to determine t_(1/2)values and subsequently, the intrinsic clearance,CL_(int)=(0.693)(1/t_(1/2) (min))(g of liver/kg of body weight)(mLincubation/mg of microsomal protein)(45 mg of microsomal protein/g ofliver weight).

Example 44: Assay Results of Tested Compounds

The following applies to Table 4 below.

Assay 1 is the biochemical MEK IC₅₀ (nM) assay as described in Example38. A1 indicates an IC₅₀ of less than or equal to 150 nM, and B1indicates an IC₅₀ of greater than 150 nM and less than or equal to 1.5μM.

Assay 2 is the A375 (BRAF) GI₅₀ (nM) cell-based assay as described inExample 41. A2 indicates an IC₅₀ of less than or equal to 500 nM, and B2indicates an IC₅₀ of greater than 500 nM and less than or equal to 1.5μM.

Assay 3 is the HCT116 (Kras) GI₅₀ (nM) assay as described in Example 41.A3 indicates an IC₅₀ of less than or equal to 750 nM, and B3 indicatesan IC₅₀ of greater than 750 nM and less than or equal to 2 μM.

Assay 4 is the liver S9 half-life stability assay as described inExample 42. A4 indicates a half life of greater than 50 min and lessthan or equal to 200 min and B4 indicates a half life of less than orequal to 50 minutes.

TABLE 4 Assay Results of Tested Compounds Comp. Assay No. 1 2 3 4 1.007B1 B2 B3 B4 1.008 A1 B2 B3 B4 1.009 B1 — — B4 1.010 A1 A2 A3 A4 1.030 A1A2 A3 A4 1.031 A1 A2 B3 B4

Example 45: In Vivo Model

Study Procedures: A topical formulation of a compound described hereinalong with a topical formulation of vehicle are applied to the skin ofnude mice in duplicate. Skin is biopsied at discrete time intervals andbisected with half snap frozen in liquid nitrogen and half formalinfixed and paraffin embedded. Protein is isolated for Western blotanalysis for p-ERK levels. p-ERK immunostaining is performed of FFPEsections for cell-specific analysis of p-ERK levels. Additional analysisincludes H&E staining to investigate skin integrity.

A compound is assessed in suppressing p-ERK, a downstream biomarker ofRAS/MAPK signaling in murine skin. In addition, proliferation of murineskin, apoptosis in murine skin, and histologic integrity of murine skinare also assessed.

Mice: 8 week old 129 mice obtained from Jackson laboratories are shavedprior to start of study. Approximately 21 mice were used for study. Acompound is applied to the hairless dorsal skin of the mouse and at 12hour intervals and skin biopsies are obtained prior to treatment, 24hours, 72 hours and at 96 hours using 6 mm punch biopsies.

Western Blot analysis: For immunoblotting, epidermal skin is snap frozenin liquid nitrogen immediately afterbiopsy. The epidermis is lysed inlysis buffer and run on Western blots. Antibodies used forimmunoblotting include rabbit anti-phospho-p44/42 MAPK (1:3000, CellSignaling) and rabbit anti-p44/42 MAPK (1:3000, Cell Signaling), mouseanti-actin (1:5,000, Sigma-Aldrich), donkey anti-mouse IgG conjugated tohorseradish peroxidase (HRP; 1:40,000, Amersham Biosciences) and goatanti-rabbit IgG conjugated HRP (1:40,000, Jackson ImmunoResearch).

Immunohistochemistry: Immunohistochemistry is performed on 5 μm paraffinsections. Antigen retrival is accomplished with enzyme treatment(1:1000) using standard protocols. Antibodies used are rabbit p-ERK(Cell Signaling, 4307S, 1:100). Bond Polymer Refine anti-rabbit HRPDetection (Leica Biosystems) is used according to manufacturer'sprotocol. Sections are then counterstained with hematoxylin, dehydratedand film coverslipped using a TissueTek-Prisma and Coverslipper(Sakura).

Histologic analysis: H&E is performed on 5 μM paraffin sections andtissue is examined to assess for cellular toxicity, inflammation orother changes in the integrity of murine skin.

Exogenous RAS activation in murine skin: The experiments are to beconducted in untreated murine skin. Alternatively, skin is pre-treatedwith TPA to enhance p-ERK levels. TPA-induced RAS/MAPK activation isperformed with 96 hours of 12.5 uG TPA in 100 μL acetone to the skin ofnude mice. Studies are performed 48 hours after TPA exposure.

T-test is used to assess differences in p-ERK and Ki-67 in samplestreated with topical MEK1 inhibitors compared to vehicle control.

Example 46: In Vivo Model

A compound described herein is tested in a mouse model of NF1, e.g.,genetically modified mouse model of NF1, a human dermal neurofibromaxenograft to nude mouse model or both. For example, methods using theNf1^(flox/flox); Dhh-Cre mouse model described in Jousma et al. Pediatr.Blood Cancer 62: 1709-1716, 2015 are used in this study. Magneticresonance imaging (MRI) and volumetric measurements is used to measuretumor volumes.

Example 47: Human Dermal Neurofibroma Explant Protocol

Dermal neurofibromas (or cutaneous neurofibromas) are benign tumorswhich develop in individuals affected with Neurofibromatosis-1 (NF1), arare genetic disease caused by mutations in the NF1 gene, leading todownstream activation of the RAS/MAPK pathway. Recent studies havedemonstrated that inhibition of MEK1 using systemic MEK inhibitors cansuppress neurofibromas and other NF-1 related tumors in murine models.See, for example, New Engl J Med 2016, 375; 26; J Clin Invest. 2013,123(1), 340-347; and Pediatr Blood Cancer 2015, 62(10), 1709-1716. Thisstudy establishes an in vitro neurofibroma explant model.

Study Objectives: The primary objective was to assess the efficacy of acompound described herein or a topically-formulated compound describedherein in suppressing p-ERK, a downstream biomarker of RAS/MAPKsignaling in neurofibroma explants. The secondary objectives was toassess permeability (where the compound was applied topically) ofneurofibroma explants treated with a compound described herein.

Protocol-1:

Sample Collection and Eligibility: Primary dermal neurofibromas orcutaneous neurofibromas are obtained from patients with clinical orgenetic diagnoses of NF1. Discarded human neurofibromas samples areobtained from the Stanford Surgery Clinic, using an approved humansubjects protocol (Stanford IRB #18325). Specimens are identified underthe direction of the Principal Investigator and placed in cellproliferation media (DMEM/F12 containing penicillin/streptomycin (0.1%);fungizone (40 μg/mL); B27 (without vitamin A).

Patients have the following data to be enrolled in the study: Patient isolder than 18 years of age; patient is not undergoing chemotherapytreatment at time of biopsy; and patients met clinical and/or geneticdiagnosis of NF1 based on presence of two of the following:

-   -   1. Six or more café-au-lait macules over 5 mm in diameter in        prepubertal individuals and over 15 mm in greatest diameter in        postpubertal individuals.    -   2. Two or more neurofibromas of any type or one plexiform        neurofibroma.    -   3. Freckling in the axillary or inguinal regions.    -   4. Two or more Lisch nodules (iris hamartomas).    -   5. Optic glioma.    -   6. A distinctive osseous lesion such as sphenoid dysplasia or        thinning of long bone cortex, with or without pseudarthrosis.    -   7. First-degree relative (parent, sibling, or offspring) with        NF-1 by the above criteria.

Study procedures: Samples are primary, untreated neurofibromas of atleast 6 mm in size; samples are excised by a shave, punch biopsy orelliptical excision; samples have a histologic diagnosis of dermalneurofibroma or cutaneous neurofibroma. Specimens are identified underthe direction of the Principal Investigator

Specimens are chopped into 2 mm fragments and placed in 24-well platescontaining cell proliferation media (DMEM/F12 containingpenicillin/streptomycin (0.1%); fungizone (40 μg/mL); B27 (withoutvitamin A) and submerged in media with drug. For topical gelapplication, samples are placed in 96 well plates with epidermal surfaceexposed to air.

Western Blot analysis: For immunoblotting, total skin biopsies are lysedin lysis buffer and run on Western blots. Antibodies used forimmunoblotting include rabbit anti-phospho-p44/42 MAPK (1:3000, CellSignaling) and rabbit anti-p44/42 MAPK (1:3000, Cell Signaling), rabbitanti-phospho-Mek1/2 (1:3000, Cell Signaling), mouse anti-actin (1:5000,Sigma-Aldrich), donkey anti-mouse IgG conjugated to horseradishperoxidase (HRP; 1:40,000, Amersham Biosciences) and goat anti-rabbitIgG conjugated HRP (1:40,000, Jackson ImmunoResearch).

Immunohistochemistry: Immunohistochemistry is performed on 5 μm paraffinsections. Antigen retrieval is accomplished with enzyme treatment(1:1000) using standard protocols. Antibodies used are rabbit p-ERK(Cell Signaling, 4307S, 1:100). Bond Polymer Refine anti-rabbit HRPDetection (Leica Biosystems) is used according to manufacturer'sprotocol. Sections are then counterstained with hematoxylin, dehydratedand film coverslipped using a TissueTek-Prisma and Coverslipper(Sakura).

Data Analysis: Semi-quantative Western blot is used to assessdifferences in p-ERK in samples treated with a compound described hereincompared to vehicle control.

Study Management: The study is conducted with oversight from an IRB withpatient informed consent and HIPAA authorization.

Protocol-2:

Explant Protocol: Human cutaneous neurofibroma explant samples werecollected in DMEM/F-12 (Thermo Fisher, Cat #11320033) supplemented with1×B27 supplement (Thermo Fisher, Cat #17504044), 2.5 μg/ml ofAmphotericin B (Thermo Fisher, Cat #15290018), and 50 units/ml ofPenicillin-50 μg/ml of Streptomycin (Thermo Fisher, Cat #15070063) andincubated in the same medium for subsequent treatment. The specimen wascut into small cubes containing both the epidermis and dermis. Thetissues were partially submerged in the medium in 384-well plate withthe epidermis exposed to the air.

The tissues were fully submerged in the medium in 48-well plate and 5 μlof compounds dissolved in DMSO were added to 200 μl of the medium ineach well. After 4 h incubation at 37° C. and 5% CO₂, the tissues wereharvested and half of the specimen was flash frozen in liquid nitrogenfor Western Blot analysis. The other half of the specimen was fixed for24 hours in 10% formalin and then transferred to 70% ethanol for WesternBlot analysis.

Skin samples were thawed on ice and weighed. 10 volume (10 μl for eachmg of tissue) of lysis buffer (RIPA buffer+0.5 mM EDTA+1×Halt proteaseand phosphatase inhibitor cocktail) was then added to each sample. Thesamples were cut into smaller pieces and homogenized with asonicatingprobe on ice. The homogenized samples were centrifuged at 12,000 rpm 4°C. for 10 minutes. Supernatant was transferred to a new tube and storedat −80° C. until Western Blot analysis.

Western Blot analysis: The lysate was thawed on ice and proteinconcentration was determined by the BCA protein assay kit using bovineserum albumin (BSA) as standards. All samples were diluted with lysisbuffer to reach the same final concentration. 10-20 μg of total proteinwas loaded to each well and separated on a NuPAGE 4-12% Bis-Tris gel(Thermo Fisher) in 1×NuPAGE MES SDS running buffer (Thermo Fisher, Cat#NP0002). Proteins were then transferred to a PVDF membrane. Themembrane was then blocked for 1 hour in 1×TBST (Tris bufferedsaline+0.1% Tween 20) with either 5% non-fat milk (for total ERK) or 5%BSA (for phospho-ERK and α-tubulin). The following primary antibodieswere used (diluted in the same blocking solution): monoclonal rabbitanti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (CellSignaling, Cat #4370L) at 1:3000, monoclonal rabbit anti-p44/42 MAPK(Erk1/2) antibody (Cell Signaling, Cat #4695S) at 1:3000, monoclonalmouse anti-α-tubulin (DM1A) antibody (Cell Signaling, Cat #3873S) at1:3000-1:4000. The membrane was incubated with the primary antibodiesovernight at 4° C. followed by three washes with 1×TBST. Secondaryantibodies goat anti-rabbit IgG (H+L), HRP (Thermo Fisher, Cat #31460)and peroxidase-conjugated affinipure goat anti-mouse IgG (H+L) (JacksonImmuno Research, Cat #115-035-062) were diluted in 1×TBST with 2%non-fat milk for total ERK and or 2% BSA for phospho-ERK and α-tubulinwith the same concentration of primary antibodies and incubated for 1-3hours at room temperature. After three washes with 1×TBST, the blotswere developed with either WesternBright ECL HRP substrate (Advansta,Cat #K12045-D50).

Immunohistochemistry: Antigen retrieval was accomplished with enzymatictreatment. Sections were blocked with 10% normal goat serum andsubsequently incubated in phospho-p44/42 MAPK (Erk1/2) rabbit monoclonalantibody (Cell Signaling) or mouse anti-Ki-67 (Pharmingen) at 1:100dilution for 60 minutes at room temperature. Detection was achieved witha peroxidase-conjugated anti-rabbit system (Leica Biosystem).

Data Analysis: Semi-quantative Western blot was used to assessdifferences in p-ERK in samples treated with a compound described hereincompared to vehicle control.

FIG. 4 demonstrates suppression of pERK in human cutaneous neurofibroma(cNF) explants after treatment for 4 hours with Compound 1.030 atconcentrations of 5 nM, 50 nM, and 500 nM.

FIG. 5 demonstrates suppression of pERK in human cutaneous neurofibroma(cNF) explants after treatment with Compound 1.030 at a concentration of500 nM.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A compound of formula (I):

or a stereoisomer, mixture of stereoisomers, and/or a pharmaceuticallyacceptable salt thereof, wherein: X is —CR^(3b) or N; R¹ is —OR⁴,—NR⁵R^(5a), —N(OR^(5b))R^(5a), or a N-linked heterocycloalkyl which isunsubstituted or substituted with one or two R⁶; R² is halo, C₁-C₆alkyl, —S—C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R^(2a) is halo or C₁-C₆ alkyl; R³, R^(3a), and R^(3b) are eachindependently hydrogen, halo, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; R⁴, R⁵, and R^(5b) are each independently hydrogen, C₁-C₆alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl-C₁-C₆ alkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxy-C₁-C₆ alkyl, amino-C₁-C₆ alkyl, C₁-C₆alkylamino-C₁-C₆ alkyl, di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl,heterocycloalkyl, heterocycloalkyl-C₁-C₆ alkyl, or R⁷—C(O)—C₁-C₆ alkyl,wherein each of the C₃-C₈ cycloalkyl and heterocycloalkyl groups isunsubstituted or substituted with one to six R⁶; R^(5a) is hydrogen orC₁-C₆ alkyl; each R⁶ is independently halo, hydroxy, oxo, C₁-C₆ alkyl,C₃-C₈ cycloalkyl, C₁-C₆ alkoxy, C₁-C₆-hydroxyalkyl, C₁-C₆ haloalkyl,amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino, amino-C₁-C₆ alkyl, C₁-C₆alkylamino-C₁-C₆ alkyl, or di-(C₁-C₆ alkyl)amino-C₁-C₆ alkyl; R⁷ ishydroxy, C₁-C₆ alkoxy, amino, C₁-C₆ alkylamino, di-(C₁-C₆ alkyl)amino,hydroxyamino, or N—C₁-C₆ alkyl hydroxyamino; and provided that thecompound is not ethyl6-cyano-2-((2-fluoro-4-iodophenyl)amino)-5-methylnicotinate.
 2. Thecompound of claim 1, having formula Ia:


3. The compound of claim 1, having formula Ib:


4. The compound of claim 1, wherein R³ is hydrogen; R^(3a) is hydrogen;and R^(3b), when present, is hydrogen or halo.
 5. The compound of claim4, wherein R^(3b) is fluoro.
 6. The compound of claim 1, wherein R² ishalo.
 7. The compound of claim 1, wherein R² is iodo.
 8. The compound ofclaim 1, wherein R^(2a) is halo.
 9. The compound of claim 1, whereinR^(2a) is fluoro.
 10. The compound of claim 1, wherein R¹ is—N(OR^(5b))R^(5a) and R^(5b) is hydrogen, C₁-C₆ alkyl, C₃-C₈ cycloalkyl,C₃-C₈ cycloalkyl-C₁-C₆ alkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy-C₁-C₆alkyl, amino-C₁-C₆ alkyl, C₁-C₆ alkylamino-C₁-C₆ alkyl, di-(C₁-C₆alkyl)amino-C₁-C₆ alkyl, heterocycloalkyl, or heterocycloalkyl-C₁-C₆alkyl, wherein each of the C₃-C₈ cycloalkyl and heterocycloalkyl groupsis unsubstituted or substituted with one to six R⁶ and each R⁶ isindependently hydroxy or C₁-C₆ alkyl.
 11. The compound of claim 1,wherein R¹ is —N(OR^(5b))R^(5a) and R^(5b) is C₃-C₈ cycloalkyl-C₁-C₆alkyl, wherein the C₃-C₈ cycloalkyl group is unsubstituted orsubstituted with one to six R⁶ and each R⁶ is independently hydroxy orC₁-C₆ alkyl.
 12. The compound of claim 1, wherein R¹ is—N(OR^(5b))R^(5a) and R^(5b) is C₁-C₆ hydroxyalkyl.
 13. The compound ofclaim 1, wherein —OR^(5b) is selected from the group consisting of —OH,


14. The compound of claim 1, wherein R¹ is —NR⁵R^(5a) or—N(OR^(5b))R^(5a), and R^(5a) is hydrogen.
 15. The compound of claim 1,selected from the group consisting of:

or a stereoisomer, mixture of stereoisomers, and/or a pharmaceuticallyacceptable salt thereof.
 16. The compound of claim 1, represented by theformula:

or a pharmaceutically acceptable salt thereof.
 17. The compound of claim1, represented by the formula:

or a pharmaceutically acceptable salt thereof
 18. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 19. A method of treating a MEK-inhibitor responsivedermal disorder or a MEK-mediated dermal disorder comprisingadministering a therapeutically effective amount of a compound of claim1, to a patient in need thereof, wherein the MEK-inhibitor responsivedermal disorder or MEK-mediated dermal disorder is selected from thegroup consisting of dermal rasopathy, neurofibromatosis type 1, dermalneurofibroma, subdermal neurofibroma, and superficial plexiformneurofibroma.
 20. The method of claim 19, wherein the dermal rasopathyis selected from the group consisting of psoriasis, keratocanthoma (KA),hyperkeratosis, papilloma, Noonan syndrome (NS), cardiofaciocutaneoussyndrome (CFC), Costello syndrome (faciocutaneoskeletal syndrome or FCSsyndrome), oculoectodermal syndrome, cafe au lait spots, and Multiplelentigines syndrome (formerly called Leopard syndrome).
 21. The methodof claim 19, wherein the compound or composition is administeredtopically, transdermally, or intralesionally.